Polynucleotides encoding anti-GD2 antibodies

ABSTRACT

Described herein are antibodies that specifically bind ganglioside GD2. Also described are nucleotides encoding such antibodies, cells expressing such antibodies, methods of use for such antibodies, and methods for using the antibodies to treat diseases associated with ganglioside GD2. In addition, tissue culture media supplements are described as are methods of use for the supplements. Described herein are albumin-ganglioside conjugates and corresponding methods for producing such conjugates. Methods of purifying or isolating antibodies are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/077,041, filed Jun. 30, 2008, and of U.S. Provisional Application No.61/097,034, filed Sep. 15, 2008, both of which are incorporated byreference herein.

TECHNICAL FIELD

The invention generally relates to the field of immunotherapeuticagents. More specifically, the invention relates to monoclonalantibodies that bind gangliosides and methods for using such antibodiesto treat subjects in need of such treatment. Also provided are methodsfor using the described antibodies for diagnostic and therapeuticpurposes. In addition, tissue culture media compositions and methodsthat relate to culturing cells expressing the described antibodies, aswell as methods for purifying or isolating the described antibodies arealso provided.

BACKGROUND

Cancer is the second leading cause of death in the United States. TheNational Cancer Institute (NCI) of the U.S. National Institutes ofHealth (NIH) estimates that over 500,000 individuals will die fromcancer and over 1.4 million individuals will be diagnosed with cancer in2008. There is a global research effort to develop methods of treatmentfor cancer and to enhance the general understanding of how to preventcancer, with countries like the United Kingdom, the Unites States, andothers making cancer-related research a top priority. For example, theNIH has earmarked over 5 billion dollars in funding for cancer-relatedresearch for the 2008 fiscal year, almost twice the amount of fundingprovided for other serious diseases, such as HIV and heart disease.

There are over 100 different types of cancer. Despite the wide varietyof cancers, most cancer-related deaths are caused by a few commoncancers, such as lung cancer, colorectal cancer, and breast cancer. TheNCI estimates that these three forms of cancer will cause over 250,000deaths in 2008. While not as deadly as those cancers mentionedpreviously, skin cancer represents about half of all newly diagnosedcancers in the U.S., making it the most common type of cancer. Of thevarious types of skin cancer, melanoma is the rarest and most deadly.The American Cancer Society estimates that while melanoma accounts foronly 4% of all diagnosed skin cancers in the U.S., it causes 79% of allskin cancer-related deaths.

Successful cancer treatment is often attributed to early diagnosis andtreatment. A powerful method of diagnosing cancer is detecting tumormarkers known to be associated with cancer. Tumor markers aresubstances, often proteins, which are produced by tumor cells, or othercells in the body in response to cancer. Tumor markers can be found inthe blood, urine, on the surface of tumor cells, or on (or in) other,non-cancerous cells and tissues. Gangliosides have been identified asone type of marker associated with many tumors (Hakomori, 99 Proc. Natl.Acad. Sci. U.S.A. 10718 (2002)).

Gangliosides are glycosphingolipids with at least one sugar-linkedsialic acid. These compounds are components of cell plasma membranes andare thought to play a central role in a variety of biological functions,such as cell growth, cell differentiation, cell signaling, and servingas receptors for microbial toxins (Jacques, et al. 4 Org. Biomol. Chem.142 (2006)). Over 40 different gangliosides have been identified;however, a certain subset of these, GM3, GM2, GD3, and GD2, are commonlyover-expressed by tumor cells (Id). Furthermore, gangliosides arereported to be highly immunogenic in humans. The combination of highimmunogenicity and over-expression by tumor cells makes gangliosides apotential target for cancer therapeutics.

Ganglioside-specific monoclonal antibodies have been developed and, insome cases, examined for efficacy in humans (See generally, Azuma etal., 13 Clin. Cancer Res. 2745 (2007); Irie, et al., 53 Cancer Immunol.Immunother. 110 (2004); Irie and Morton, 83 Proc. Natl. Acad. Sci.U.S.A. 8694 (1986)). One of the earliest reports concerning a human,GD2-specific antibody was by Cahan, et al. (79 Proc. Natl. Acad. Sci.U.S.A. 7629 (1982)). Immunotherapy studies have shown that highersurvival rates correlate with ganglioside-specific IgM, rather than IgG,for patients vaccinated with a melanoma vaccine (Jones et al., 66 J.Natl. Cancer Inst. 249 (1981)). Consistent with this finding, humanstudies involving IgM antibodies to gangliosides GD2 and GD3 haveprovided encouraging results for the use of such antibodies as apossible treatment for melanoma (Irie, et al., 53 Cancer Immunol.Immunother. 110; Irie and Morton, 83 Proc. Natl. Acad. Sci. U.S.A. 8694(1986)).

IgM is one of five human antibody isotypes; IgG, IgA, IgE, and IgD arethe others. IgM is typically the first antibody produced in a humoralimmune response because the position of IgM heavy chain constant genesallows it to be produced without isotype switching (Charles A. Janeway,et al. Immunobiology 9-12 (5^(th) ed. 2001)). Because IgM is oftenproduced before genetic maturation occurs, this class of antibodyisotype typically has lower affinity for a given antigen than otherisotypes (Id). IgM compensates for low affinity by forming polymers,which increase the avidity of the antibody molecule (Id). Polymeric IgMusually forms as a pentamer associated with J-chain (a ˜15 kD moleculethat promotes antibody polymerization); however, it can also polymerizeas a pentamer or hexamer in the absence of J-chain (Id. at 4-19). Insome instances, the polymeric state of IgM allows it to mediate highlyeffective activation of the complement pathway in the presence ofpathogens. For example, J-chain-containing pentameric IgM is not usuallyeffective in activating complement unless it undergoes a structuralchange when bound to an antigen (Id. at 9-17). In contrast, J-chain-freeor hexameric IgM has been shown to activate complement up to 100-foldbetter than pentameric IgM (Weirsma et al., 160 J. Immunol. 5979(1998)).

Cancer is a global health problem. Though progress has been made intreating various forms of this disease, improved therapeutics areneeded. Immunotherapeutics are thought to have great potential forcancer therapy. An IgM antibody specific for an antigen expressed bycancer cells may prove to be an effective cancer therapeutic.

SUMMARY

Described herein are isolated antibodies and antigen-binding fragmentsthat specifically bind to ganglioside GD2. In some embodiments, theantibodies or antigen-binding fragments are IgMs. In some embodiments,the antibodies or antigen-binding fragments are pentameric or hexameric.While the antibodies or antigen-binding fragments may be human,humanized, or chimeric, the antibodies or antigen-binding fragments arepreferably human. The antibodies or antigen-binding fragments mayinclude a heavy chain having an amino acid sequence substantially thesame as or identical to SEQ ID NO: 40 and a light chain having an aminoacid sequence substantially the same as or identical to SEQ ID NO: 42.In some embodiments, antibodies or antigen-binding fragments may includea heavy chain CDR1 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 10. In some embodiments, antibodies orantigen-binding fragments may include a heavy chain CDR2 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 11. Insome embodiments, antibodies or antigen-binding fragments may include aheavy chain CDR3 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 12. In some embodiments, antibodies orantigen-binding fragments may include a light chain CDR1 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 26. Insome embodiments, antibodies or antigen-binding fragments may include alight chain CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 27. In some embodiments, antibodies orantigen-binding fragments may include a light chain CDR3 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 28. Theantibodies or antigen-binding fragments may include a heavy chain havinga CDR1 amino acid sequence substantially the same as, or identical to,SEQ ID NO: 10; a CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 11; and a CDR3 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 12. Theantibodies or antigen-binding fragments may include a light chain havinga CDR1 amino acid sequence substantially the same as, or identical to,SEQ ID NO: 26; a CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 27; and a CDR3 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 28. Theantibodies or antigen-binding fragments may include a heavy chain havinga CDR1 amino acid sequence substantially the same as, or identical to,SEQ ID NO: 10; a CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 11; and a CDR3 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 12, and also havea light chain having a CDR1 amino acid sequence substantially the sameas, or identical to, SEQ ID NO: 26; a CDR2 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 27; and a CDR3amino acid sequence substantially the same as, or identical to, SEQ IDNO: 28.

The antibodies or antigen-binding fragments may include a heavy chainFWR1 amino acid sequence substantially the same as, or identical to, SEQID NO: 13. In some embodiments, the antibodies or antigen-bindingfragments include a heavy chain FWR2 amino acid sequence substantiallythe same as, or identical to, SEQ ID NO: 14. In some embodiments, theantibodies or antigen-binding fragments include a heavy chain FWR3 aminoacid sequence substantially the same as, or identical to, SEQ ID NO: 15.In some embodiments, the antibodies or antigen-binding fragments includea light chain FWR1 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 29. In some embodiments, the antibodies orantigen-binding fragments include a light chain FWR2 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 30. In someembodiments, the antibodies or antigen-binding fragments include a lightchain FWR3 amino acid sequence substantially the same as, or identicalto, SEQ ID NO: 31. In some embodiments, the antibodies orantigen-binding fragments include a heavy chain having a FWR1 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 13; aFWR2 amino acid sequence substantially the same as, or identical to, SEQID NO: 14; and a FWR3 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 15. In some embodiments, the antibodies orantigen-binding fragments include a light chain having a FWR1 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 29; aFWR2 amino acid sequence substantially the same as, or identical to, SEQID NO: 30; and a FWR3 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 31. In some embodiments the antibodies orantigen-binding fragments may include a heavy chain and a light chain,wherein the heavy chain includes a FWR1 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 13; a FWR2 aminoacid sequence substantially the same as, or identical to, SEQ ID NO: 14;and a FWR3 amino acid sequence substantially the same as, or identicalto, SEQ ID NO: 15; and the light chain includes a FWR1 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 29; aFWR2 amino acid sequence substantially the same as, or identical to, SEQID NO: 30; and a FWR3 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 31.

In some embodiments, the antibodies or antigen-binding fragments includea heavy chain having a CDR1 amino acid sequence substantially the sameas, or identical to, SEQ ID NO: 10; a CDR2 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 11; and a CDR3amino acid sequence substantially the same as, or identical to, SEQ IDNO: 12; a FWR1 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 13; a FWR2 amino acid sequence substantiallythe same as, or identical to, SEQ ID NO: 14; and a FWR3 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 15. Insome embodiments, the antibodies or antigen-binding fragments include alight chain having a CDR1 amino acid sequence substantially the same as,or identical to, SEQ ID NO: 26; a CDR2 amino acid sequence substantiallythe same as, or identical to, SEQ ID NO: 27; and a CDR3 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 28; aFWR1 amino acid sequence substantially the same as, or identical to, SEQID NO: 29; a FWR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 30; and a FWR3 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 31. In someembodiments, the antibodies or antigen-binding fragments have a heavyand a light chain, wherein the heavy chain has a CDR1 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 10; aCDR2 amino acid sequence substantially the same as, or identical to, SEQID NO: 11; and a CDR3 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 12; a FWR1 amino acid sequence substantiallythe same as, or identical to, SEQ ID NO: 13; a FWR2 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 14; and a FWR3amino acid sequence substantially the same as, or identical to, SEQ IDNO: 15; and the light chain has a CDR1 amino acid sequence substantiallythe same as, or identical to, SEQ ID NO: 26; a CDR2 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 27; and a CDR3amino acid sequence substantially the same as, or identical to, SEQ IDNO: 28; a FWR1 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 29; a FWR2 amino acid sequence substantiallythe same as, or identical to, SEQ ID NO: 30; and a FWR3 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 31.Antigen-binding arrangements of CDRs and FWRs may also be engineeredusing antibody-like proteins as CDR scaffolding. Such engineeredantigen-binding proteins are within the scope of the disclosure.

The described antibodies or antigen-binding fragments may include aheavy chain that has an amino acid sequence substantially the same as,or identical to, SEQ ID NO: 40. In some embodiments, a polynucleotidesubstantially the same as, or identical to, SEQ ID NO: 39 may encodethis heavy chain amino acid sequence. The described antibodies orantigen-binding fragments may include a light chain that has an aminoacid sequence substantially the same as, or identical to, SEQ ID NO: 42.In some embodiments, a polynucleotide substantially the same as, oridentical to, SEQ ID NO: 41 may encode this light chain amino acidsequence. The described antibodies or antigen-binding fragments mayinclude a heavy and a light chain, wherein the heavy chain has an aminoacid sequence substantially the same as, or identical to, SEQ ID NO: 40,and the light chain has an amino acid sequence substantially the sameas, or identical to, SEQ ID NO: 42.

In some embodiments, the antibodies are produced by antibody-producingcells deposited with the Amer. Type Cult. Coll. (10801 University Blvd.,Manassas, Va. 20110-2209) on Jul. 16, 2008 and have been assignedAccession No. PTA-9376. In some embodiments, the antibodies, orantigen-binding fragments thereof, have the binding affinity for GD2 ofthe antibodies produced by the deposited antibody-producing cells. Insome embodiments, the disclosed antibodies, or antigen-binding fragmentsthereof, comprise the heavy and light chain CDRs of the antibodiesproduced by the deposited antibody-producing cells. In some embodiments,the antibodies, or antigen-binding fragments thereof, comprise the heavyand light chain variable regions of the antibodies produced by thedeposited antibody-producing cells. In some embodiments, the antibodies,or antigen-binding fragments thereof, exhibit substantially the same, orgreater, level of complement-dependent cytotoxic activity as antibodiesproduced by the deposited antibody-producing cells. In addition,antibodies, or antigen-binding fragments thereof, that compete with theantibodies produced by the deposited cells for binding to GD2 arecontemplated to be within the scope of the antibodies described herein.For instance, an antibody may compete with another antibody, or anantigen-binding fragment, if it prevents or hinders the other antibodyfrom binding to an antigenic site to which the antibody would otherwisebind in the absence of the competing antibody.

Also disclosed are polynucleotides that encode antibodies orantigen-binding fragments that specifically bind to GD2. In someembodiments, the polynucleotides encode an antibody or antigen-bindingfragment thereof having a heavy chain CDR1 sequence substantially thesame as, or identical to, SEQ ID NO: 10, for example SEQ ID NO: 2. Insome embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain CDR2 substantiallythe same as, or identical to, SEQ ID NO: 11, for example SEQ ID NO: 3.In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain CDR3 substantiallythe same as, or identical to, SEQ ID NO: 12, for example SEQ ID NO: 4.In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a light chain CDR1 substantiallythe same as, or identical to, SEQ ID NO: 26, for example SEQ ID NO: 18.In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a light chain CDR2 substantiallythe same as, or identical to, SEQ ID NO: 27, for example SEQ ID NO: 19.In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a light chain CDR3 substantiallythe same as, or identical to, SEQ ID NO: 28, for example SEQ ID NO: 20.The polynucleotides may encode an antibody or antigen-binding fragmentthereof having a heavy chain with a CDR1 substantially the same as, oridentical to, SEQ ID NO: 10, for example SEQ ID NO: 2; a CDR2substantially the same as, or identical to, SEQ ID NO: 11, for exampleSEQ ID NO: 3; and a CDR3 substantially the same as, or identical to, SEQID NO: 12, for example SEQ ID NO: 4. The polynucleotides may encode anantibody or antigen-binding fragment thereof having a light chain CDR1substantially the same as, or identical to, SEQ ID NO: 26, for exampleSEQ ID NO: 18; a CDR2 substantially the same as, or identical to, SEQ IDNO: 27, for example SEQ ID NO: 19; and a CDR3 substantially the same as,or identical to, SEQ ID NO: 28, for example SEQ ID NO: 20. Thepolynucleotides may encode an antibody or antigen-binding fragmentthereof having a heavy chain CDR1 substantially the same as, oridentical to, SEQ ID NO: 10, for example SEQ ID NO: 2; a CDR2 encoded bya nucleotide sequence substantially the same as, or identical to, SEQ IDNO: 11, for example SEQ ID NO: 3; and a CDR3 encoded by a nucleotidesequence substantially the same as, or identical to, SEQ ID NO: 12, forexample SEQ ID NO: 4; and a light chain CDR1 substantially the same as,or identical to, SEQ ID NO: 26, for example SEQ ID NO: 18; a CDR2substantially the same as, or identical to, SEQ ID NO: 27, for exampleSEQ ID NO: 19; and a CDR3 substantially the same as, or identical to,SEQ ID NO: 28, for example SEQ ID NO: 20. Antigen-binding arrangementsof CDRs may also be engineered using antibody-like proteins as CDRscaffolding. Such engineered antigen-binding proteins are within thescope of the disclosure.

In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain FWR1 substantiallythe same as, or identical to, SEQ ID NO: 13, for example SEQ ID NO: 5.In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain FWR2 substantiallythe same as, or identical to, SEQ ID NO: 14, for example SEQ ID NO: 6.In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain FWR3 substantiallythe same as, or identical to, SEQ ID NO: 15, for example SEQ ID NO: 7.In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a light chain FWR1 substantiallythe same as, or identical to, SEQ ID NO: 29, for example SEQ ID NO: 21.In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a light chain FWR2 substantiallythe same as, or identical to, SEQ ID NO: 30, for example SEQ ID NO: 22.In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a light chain FWR3 substantiallythe same as, or identical to, SEQ ID NO: 31, for example SEQ ID NO: 23.In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain FWR1 substantiallythe same as, or identical to, SEQ ID NO: 13, for example SEQ ID NO: 5; aFWR2 substantially the same as, or identical to, SEQ ID NO: 14, forexample SEQ ID NO: 6; and a FWR3 substantially the same as, or identicalto, SEQ ID NO: 15, for example SEQ ID NO: 7. In some embodiments, thepolynucleotides encode an antibody or antigen-binding fragment thereofhaving a light FWR1 substantially the same as, or identical to, SEQ IDNO: 29, for example SEQ ID NO: 21; a FWR2 substantially the same as, oridentical to, SEQ ID NO: 30, for example SEQ ID NO: 22; and a FWR3substantially the same as, or identical to, SEQ ID NO: 31, for exampleSEQ ID NO: 23. In some embodiments, the polynucleotides encode anantibody or antigen-binding fragment thereof having a heavy chain and alight chain, wherein a heavy chain FWR1 is substantially the same as, oridentical to, SEQ ID NO: 13, for example SEQ ID NO: 5; a heavy chainFWR2 is substantially the same as, or identical to, SEQ ID NO: 14, forexample SEQ ID NO: 6; and a heavy chain FWR3 is substantially the sameas, or identical to, SEQ ID NO: 15, for example SEQ ID NO: 7; and alight chain FWR1 is substantially the same as, or identical to, SEQ IDNO: 29, for example SEQ ID NO: 21; a light chain FWR2 is substantiallythe same as, or identical to, SEQ ID NO: 30, for example SEQ ID NO: 22;and a light chain FWR3 substantially the same as, or identical to, SEQID NO: 31, for example SEQ ID NO: 23.

In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain CDR1 substantiallythe same as, or identical to, SEQ ID NO: 10, for example SEQ ID NO: 2; aheavy chain CDR2 substantially the same as, or identical to, SEQ ID NO:11, for example SEQ ID NO: 3; and a heavy chain CDR3 substantially thesame as, or identical to, SEQ ID NO: 12, for example SEQ ID NO: 4; aheavy chain FWR1 substantially the same as, or identical to, SEQ ID NO:13, for example SEQ ID NO: 5; a heavy chain FWR2 substantially the sameas, or identical to, SEQ ID NO: 14, for example SEQ ID NO: 6; and aheavy chain FWR3 substantially the same as, or identical to, SEQ ID NO:15, for example SEQ ID NO: 7. In some embodiments, the polynucleotidesencode an antibody or antigen-binding fragment thereof having a lightchain CDR1 substantially the same as, or identical to, SEQ ID NO: 26,for example SEQ ID NO: 18; a light chain CDR2 substantially the same as,or identical to, SEQ ID NO: 27, for example SEQ ID NO: 19; and a lightchain CDR3 substantially the same as, or identical to, SEQ ID NO: 28,for example SEQ ID NO: 20; a light chain FWR1 substantially the same as,or identical to, SEQ ID NO: 29, for example SEQ ID NO: 21; a light chainFWR2 substantially the same as, or identical to, SEQ ID NO: 30, forexample SEQ ID NO: 22; and a light chain FWR3 substantially the same as,or identical to, SEQ ID NO: 31, for example SEQ ID NO: 23.

In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy and a light chain,wherein the polynucleotides encode a heavy chain CDR1 substantially thesame as, or identical to, SEQ ID NO: 10, for example SEQ ID NO: 2; aheavy chain CDR2 substantially the same as, or identical to, SEQ ID NO:11, for example SEQ ID NO: 3; a heavy chain CDR3 substantially the sameas, or identical to, SEQ ID NO: 12, for example SEQ ID NO: 4; a heavychain FWR1 substantially the same as, or identical to, SEQ ID NO: 13,for example SEQ ID NO: 5; a heavy chain FWR2 substantially the same as,or identical to, SEQ ID NO: 14, for example SEQ ID NO: 6; and a heavychain FWR3 substantially the same as, or identical to, SEQ ID NO: 15,for example SEQ ID NO: 7; and a light chain CDR1 substantially the sameas, or identical to, SEQ ID NO: 26, for example SEQ ID NO: 18; a lightchain CDR2 substantially the same as, or identical to, SEQ ID NO: 27,for example SEQ ID NO: 19; a light chain CDR3 substantially the same as,or identical to, SEQ ID NO: 28, for example SEQ ID NO: 20; a light chainFWR1 substantially the same as, or identical to, SEQ ID NO: 29, forexample SEQ ID NO: 21; a light chain FWR2 substantially the same as, oridentical to, SEQ ID NO: 30, for example SEQ ID NO: 22; and a lightchain FWR3 substantially the same as, or identical to, SEQ ID NO: 31,for example SEQ ID NO: 23. Polynucleotides encoding engineeredantigen-binding proteins also are within the scope of the disclosure.

Vectors comprising the antibody- and antigen-binding fragment-encodingpolynucleotides are provided, as are cells expressing the antibodies orantigen-binding fragments that specifically bind to GD2.

Described herein are methods for treating or preventing GD2-associateddisease in a subject in need of such treatment. In some embodiments, theGD2-associated disease is cancer. In some embodiments, theGD2-associated disease is melanoma. The methods comprise administeringto the subject an antibody or antigen-binding fragment thereof thatspecifically binds to GD2 in an amount effective to treat or prevent theGD2-associated disease. In some embodiments the methods compriseadministering a pharmaceutical composition including an antibody orantigen-binding fragment thereof and a pharmaceutically acceptablecarrier. In some aspects, the antibody or antigen-binding fragmentthereof is an IgM antibody or antigen-binding fragment. In someembodiments, the described methods of treating or preventingGD2-associated disease in a subject may be carried out with an antibody,or antigen-binding fragment, that competes for binding with theGD2-specific antibodies described herein.

Described herein are methods for detecting GD2-expressing cells in vivoor in vitro. The methods may include administering to a subject anantibody or antigen-binding fragment thereof described herein thatspecifically binds to GD2 to allow for the detection or localization ina subject. In some embodiments, the described antibodies orantigen-binding fragments may be detectably labeled. Such detectablelabels may include fluorescent labels, radiolabels, biotin, and thelike. Alternatively, in some embodiments the GD2-specific antibodies orantigen-binding fragments are not labeled and, instead are detected by asecondary antibody which is detectably labeled. In some embodiments, thedescribed methods of detection may be carried out with an antibody, orantigen-binding fragment, that competes for binding with theGD2-specific antibodies and antigen-binding fragments described herein.

Described herein are methods of making and purifying or isolating IgMantibodies or antigen-binding fragments thereof. The methods includeculturing a host cell under conditions suitable to produce theantibodies or an antigen-binding fragments, recovering the antibodies orantigen-binding fragments from the cell culture, and purifying orisolating the recovered antibodies or antigen-binding fragments. Alsofeatured are methods of purifying or isolating IgM antibodies orantigen-binding fragments, including optionally washing the antibodiesor an antigen-binding fragments with a detergent, applying a solutioncomprising the antibodies or antigen-binding fragments to an affinitychromatography column, applying eluate from the affinity chromatographycolumn to a cation exchange chromatography column, and applying eluatefrom the cation exchange chromatography column to a hydroxyapatitechromatography column and recovering the eluate from the hydroxyapatitechromatography column.

Additional methods include enhancing the viability of culturedeukaryotic cells by supplementing cell culture media with compositionscomprising amino acids, sugars, and vitamins. In some embodiments thecompositions may comprise glucose, glutamate, aspartate, serine,histidine, threonine, arginine, tyrosine, cysteine, valine, methionine,tryptophan, phenyalanine, isoleucine, leucine, lysine, proline,nicotinic acid amide, pyridoxine HCl, folic acid, vitamin B-12,riboflavin, and thiamine HCl. Described herein are also methods forenhancing protein production by cells modified to produce proteins. Inone aspect, these methods include supplementing the growth media ofcells with valeric acid.

Described herein are protein conjugates of albumin, for example bovineserum albumin (BSA), and a ganglioside and methods for producing suchprotein conjugates. In one embodiment, the conjugated ganglioside may beGD2. In one embodiment, the conjugated ganglioside may be GM2. In oneembodiment, the conjugated ganglioside may be GM3. In one embodiment,albumin is conjugated to a reducing end of a carbohydrate moiety of aganglioside by reductive amination. In one embodiment, the reductiveamination is catalyzed by sodium cyanoborohydride.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows variable antigen specificity of antibodies present in thesupernatant of an Epstein-Barr Virus-transformed human lymphoblast pool(HLP) as determined by ELISA. FIG. 1A demonstrates that concentratedcell culture supernatants from the HLP, at cell passage 2 (8.1×concentrated) or 4 (14.2× concentrated), contain IgM antibodies specificfor GD2. FIG. 1B demonstrates that cell culture supernatants from theHLP contain IgM antibodies that bind gangliosides GD1a, GD2, GM2, andGM3.

FIG. 2 shows binding levels of IgM antibodies produced by the HLP cellcultures on GD2-expressing 1205LU melanoma cells, as analyzed byfluorescence activated cell sorting (FACS). FIG. 2A serves as negativecontrol, showing binding in the absence of GD2-specific primaryantibody. FIGS. 2B and 2C show binding of HLP-derived IgM obtained fromcell culture supernatants concentrated to either 8.1× or 14.2×,respectively. FIG. 2D shows binding of a murine GD2-specific IgMantibody produced by murine hybridoma HB-8568™ (ATCC #HB 8568™) andserves as a positive control.

FIG. 3 shows an antigen-specific ELISA using spent medium from hybridomaclones generated by the HLP-derived hybridomas. FIG. 3A shows bindingcharacteristics of IgM antibodies contained in media of hydridomas 3B2,5D7, and 10B4 for gangliosides GD2, GD1a, GM2, and GM3. FIG. 3B showsbinding characteristics of IgM antibodies contained in media of the 1470hydridoma subclone for gangliosides GD2, GD1a, GM2, and GM3.

FIG. 4 shows binding levels of IgM antibodies produced by hybridoma 3B2and 1470 cell cultures on GD2-expressing 1205LU melanoma cells, asanalyzed by FACS. FIG. 4A serves as negative control, showing lack ofbinding in the absence of GD2-specific primary antibody. FIG. 4B, whichserves as a positive control, shows binding of a murine GD2-specific IgMantibody produced by HB-8568™ cells. FIGS. 4C and D show binding of IgMproduced by 3B2 and 1470 hybridoma cells, respectively.

FIG. 5 shows cell death due to complement-dependent cytotoxicity (CDC)mediated by IgM obtained from hybridoma 3B2 tissue culture supernatantor non-GD2-specific human IgM (nhIgM).

FIG. 6 provides a schematic representation of the cloning of AB527antibody heavy and light chain segments.

FIG. 7 provides a schematic representation of the AB527 expressionvector.

FIG. 8 illustrates the binding activity of AB527 for the gangliosidesGD2, GM2, and GD3, as measured by surface plasmon resonance.

FIG. 9 provides a graphical representation of AB527 binding equilibriumas measured by surface plasmon resonance for differing concentrations ofantibody bound to GD2.

FIG. 10 shows the staining patterns of various gangliosides separated bythin layer chromatography. Orcinol staining shows non-specificganglioside staining, which provides a basis for comparison with AB527staining.

FIG. 11 shows flow cytometry (columns I and II) and comparative lightmicroscopy and immunofluorescence microscopy images (columns II and IV)of AB527 binding to human melanoma cell lines that express GD2 at thecell surface (cell lines: M14, M0023, M101, M18, M10-Vac, PM0496) orlack GD2 expression (GD2-negative lines: M21, M238, IMCD0023, MG1055).

FIG. 12 provides a graphical representation of a saturation bindingcurve and corresponding Scatchard plot that characterizes the binding ofAB527 to EL4 cells.

FIG. 13 shows AB527 staining of GD2 positive (M14) and negative (MG1055,RPMI7951 and JS0592) melanoma cell lines. Immunohistochemical analysiswas performed using biotinylated AB527 (FIG. 13A) or biotinylated,non-GD2-specific, control IgM (FIG. 13B).

FIG. 14 shows that M14 tumor sections are not immunoreactive withbiotinylated hIgM (14a), but do show immunoreactivity with biotinylatedAB527 (14b).

FIG. 15 shows a comparison of complement-dependent cytotoxicity (CDC) ofGD2 positive or GD2 negative cells mediated by either IgM obtained fromhybridoma AB527-HYB-3B2-3C9 or AB527 IgM.

FIG. 16 is a graphical representation of AB527-mediated CDC in thepresence or absence of complement, for human tumor cells that eitherexpress GD2 (M14) or do not express GD2 (1205LU).

FIG. 17 illustrates the relative degree of CDC activity for non-specifichuman IgM, AB527, non-specific hIgG, and isotype-switched AB527 havingan IgG1 constant region.

FIG. 18 shows the relative degree of CDC activity for AB527 antibodieswith or without J-chain (JC).

FIG. 19 shows a reducing SDS-PAGE gel of detergent-treated conditionedculture supernatant (CCS) applied to a protein A affinity chromatographycolumn (Load) and the material collected from pooled fractions afterelution by 3M MgCl₂ (Elute). Arrows point to the IgM heavy chain (˜70kD) and light chain (˜25 kD).

FIG. 20 shows a reducing SDS-PAGE gel of AB527 captured by differingamounts of MacroCap™ SP cation exchange resin. Arrows point to the IgMheavy chain (˜70 kD) and light chain (˜25 kD).

FIG. 21 shows relative amounts of various forms of AB527 IgM in theflowthrough (FIG. 21A) or eluate (FIG. 21B) collected after applicationof differing volumes of AB527 intermediate to MacroCap™ SP cationexchange resin.

FIG. 22 depicts chromatograms from size exclusion-HPLC, verifying thesize distribution of material present in the flowthrough (FIG. 22A) andelution (FIG. 22B) from the MacroCap™ SP column.

FIG. 23 provides a graphical representation of AB527 binding to theGD2-BSA conjugate.

FIG. 24 provides a graphical representation comparing AB527 binding toGD2-BSA, GM2-BSA, and GM3-BSA conjugates.

FIG. 25 shows a comparison of viable cell density over time forAB527-expressing cells cultured in media with (culture media supplement(CMS)) and without (CD CHO control) the addition of an embodiment of theCMS.

FIG. 26 shows the relative percentage of viable cells over time forAB527-expressing cells cultured in media with (CMS) and without (CD CHOcontrol) the addition of an embodiment of the CMS.

FIG. 27 shows the integral viable cell density (IVC) over time forAB527-expressing cells cultured in media with (CMS) and without (CD CHOcontrol) the addition of an embodiment of the CMS.

FIG. 28 shows the total antibody production, after 14 days in culture,for AB527-expressing cells cultured in media with (CMS) and without (CDCHO control) the addition of an embodiment of the CMS.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Various terms relating to aspects of the description are used throughoutthe specification and claims. Such terms are to be given their ordinarymeaning in the art unless otherwise indicated. Other specificallydefined terms are to be construed in a manner consistent with thedefinitions provided herein.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a cell”includes a combination of two or more cells, and the like.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of up to ±20% from the specified value, as suchvariations are appropriate to perform the disclosed methods. Unlessotherwise indicated, all numbers expressing quantities of ingredients,properties such as molecular weight, reaction conditions, and so forthused in the specification and claims are to be understood as beingmodified in all instances by the term “about.” Accordingly, unlessindicated to the contrary, the numerical parameters set forth in thefollowing specification and attached claims are approximations that mayvary depending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contain certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements.

As used herein, the term “cytotoxic” or “cytostatic” agent refers to anagent that inhibits the biological processes of a cell, or reduces theviability or proliferative potential of a cell. Cytotoxic or cytostaticagents may function in a variety of ways, for example, but not by way oflimitation, by inducing DNA damage, inducing cell cycle arrest,inhibiting DNA synthesis, inhibiting transcription, inhibitingtranslation or protein synthesis, inhibiting cell division, or inducingapoptosis. As used herein, the term “chemotherapeutic agent” refers tocytotoxic, cytostatic, and antineoplastic agents that preferentiallykill, inhibit the growth of, or inhibit the metastasis of neoplasticcells or disrupt the cell cycle of rapidly proliferating cells.Chemotherapeutic agents include, but are not limited to, syntheticcompounds, natural and recombinant bacterial toxins, natural andrecombinant fungal toxins, natural and recombinant plant toxins,fissionable nuclides, and radionuclides. Specific examples ofchemotherapeutic agents include, but are not limited to, pokeweedantiviral protein, abrin, ricin and each of their A chains, momordin,saporin, bryodin 1, bouganin, gelonin, Diphtheria toxin, Pseudomonasexotoxin, Shiga toxin, calicheamicin, maytansinoid, lead-212,bismuth-212, astatine-211, iodine-131, scandium-47, rhenium-186,rhenium-188, yttrium-90, iodine-123, iodine-124, iodine-125, bromine-77,indium-111, boron-10, actinide, altretamine, actinomycin D, plicamycin,puromycin, gramicidin D, doxorubicin, colchicine, cytochalasin B,cyclophosphamide, emetine, maytansine, amsacrine, cisplastin, etoposide,etoposide orthoquinone, teniposide, daunorubicin, gemcitabine,doxorubicin, mitoxantraone, bisanthrene, Bleomycin, methotrexate,vindesine, adriamycin, vincristine, vinblastine, BCNU, taxol, tarceva,avastin, mitomycin, 5-fluorouracil, cyclophosphamide and certaincytokines such as TNF-alpha and TNF-beta.

“Isolated” means altered “by the hand of man” from the natural state. Ifa molecule or composition occurs in nature, it has been “isolated” if ithas been changed or removed from its original environment, or both. Forexample, a polynucleotide or a polypeptide naturally present in a livingplant or animal is not “isolated,” but the same polynucleotide orpolypeptide separated from the coexisting materials of its natural stateis “isolated” as the term is employed herein.

“Polynucleotide,” synonymously referred to as “nucleic acid molecule” or“nucleic acids,” refers to any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term polynucleotide also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications may be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short nucleic acidchains, often referred to as oligonucleotides.

“Substantially the same” with respect to nucleic acid or amino acidsequences, means at least 65% identity between two or more sequences.Preferably, the term refers to at least 70% identity between two or moresequences, more preferably at least 75% identity, more preferably atleast 80% identity, more preferably at least 85% identity, morepreferably at least 90% identity, more preferably at least 91% identity,more preferably at least 92% identity, more preferably at least 93%identity, more preferably at least 94% identity, more preferably atleast 95% identity, more preferably at least 96% identity, morepreferably at least 97% identity, more preferably at least 98% identity,and more preferably at least 99% or greater identity. Such identity maybe determined using mBLAST algorithm (Altschul et al. (1990) Proc. Natl.Acad. Sci. USA 87:2264-8; Karlin and Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-7).

A “vector” is a replicon, such as plasmid, phage, cosmid, or virus inwhich another nucleic acid segment may be operably inserted so as tobring about the replication or expression of the segment.

The term “operably linked” or “operably inserted” means that theregulatory sequences necessary for expression of the coding sequence areplaced in a nucleic acid molecule in the appropriate positions relativeto the coding sequence so as to enable expression of the codingsequence. By way of example, a promoter is operably linked with a codingsequence when the promoter is capable of controlling the transcriptionor expression of that coding sequence. Coding sequences may be operablylinked to promoters or regulatory sequences in a sense or antisenseorientation. The term “operably linked” is sometimes applied to thearrangement of other transcription control elements (e.g., enhancers) inan expression vector.

A cell has been “transformed” when exogenous or heterologous nucleicacids such as DNA have been introduced inside the cell. The transformingDNA may or may not be integrated (covalently linked) into the genome ofthe cell. In prokaryotes, yeast, and mammalian cells for example, thetransforming DNA may be maintained on an episomal element such as aplasmid. With respect to eukaryotic cells, a stably transformed cell, or“stable cell” is demonstrated by the ability of the eukaryotic cell toestablish cell lines or clones comprised of a population of daughtercells containing the transforming DNA. A “clone” is a population ofcells derived from a single cell or common ancestor by mitosis. A “cellline” is a clone of a primary cell that is capable of stable growth invitro for many generations. In some examples provided herein, cells aretransformed by transfecting the cells with DNA.

“Polypeptide” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. “Polypeptide” refers to both shortchains, commonly referred to as peptides, oligopeptides or oligomers,and to longer chains, generally referred to as proteins. Polypeptidesmay contain amino acids other than the 20 gene-encoded amino acids.“Polypeptides” include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts, monographs, andresearch literature. Modifications may occur anywhere in a polypeptide,including the peptide backbone, the amino acid side-chains and the aminoor carboxyl termini. The same type of modification may be present in thesame or varying degrees at several sites in a given polypeptide. Also, agiven polypeptide may contain many types of modifications. Polypeptidesmay be branched as a result of ubiquitination, and they may be cyclic,with or without branching. Cyclic, branched and branched cyclicpolypeptides may result from natural posttranslational processes or maybe made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination (See, for instance, Proteins—Structure and MolecularProperties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork, 1993 and Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in Posttranslational CovalentModification of Proteins, B. C. Johnson, Ed., Academic Press, New York,1983; Seifter et al., Analysis for Protein Modifications and NonproteinCofactors, Meth Enzymol (1990) 182:626-646 and Rattan et al., ProteinSynthesis: Posttranslational Modifications and Aging, Ann NY Acad Sci(1992) 663:48-62).

“Biomolecules” include proteins, polypeptides, nucleic acids, lipids,monosaccharides, polysaccharides, and all fragments, analogs, homologs,conjugates, and derivatives thereof.

The terms “express” and “produce” are used synonymously herein, andrefer to the biosynthesis of a gene product. These terms encompass thetranscription of a gene into RNA. These terms also encompass translationof RNA into one or more polypeptides, and further encompass allnaturally occurring post-transcriptional and post-translationalmodifications. The expression or production of an antibody orantigen-binding fragment thereof may be within the cytoplasm of thecell, or into the extracellular milieu such as the growth medium of acell culture.

The terms “treating” or “treatment” refer to any success or indicia ofsuccess in the attenuation or amelioration of an injury, pathology orcondition, including any objective or subjective parameter such asabatement, remission, diminishing of symptoms or making the injury,pathology, or condition more tolerable to the patient, slowing in therate of degeneration or decline, making the final point of degenerationless debilitating, improving a subject's physical or mental well-being,or prolonging the length of survival. The treatment may be assessed byobjective or subjective parameters; including the results of a physicalexamination, neurological examination, or psychiatric evaluations.

“Effective amount” and “therapeutically effective amount” are usedinterchangeably herein, and refer to an amount of an antibody,antigen-binding fragment, or antibody composition, as described herein,effective to achieve a particular biological or therapeutic result suchas, but not limited to, biological or therapeutic results disclosed,described, or exemplified herein. A therapeutically effective amount ofthe antibody or antigen-binding fragment thereof may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of the antibody or antigen-binding fragmentthereof to elicit a desired response in the individual. Such results mayinclude, but are not limited to, the treatment of cancer, as determinedby any means suitable in the art.

“Pharmaceutically acceptable” refers to those properties and substanceswhich are acceptable to the patient from a pharmacological/toxicologicalpoint of view and to the manufacturing pharmaceutical chemist from aphysical/chemical point of view regarding composition, formulation,stability, patient acceptance, and bioavailability.

“Pharmaceutically acceptable carrier” refers to a medium that does notinterfere with the effectiveness of the biological activity of theactive ingredient(s) and is not toxic to the host to which it isadministered.

“Antibody” refers to all isotypes of immunoglobulins (IgG, IgA, IgE,IgM, IgD, and IgY) including various monomeric and polymeric forms ofeach isotype, unless otherwise specified.

Antigen-binding fragments are any proteinaceous structure that mayexhibit binding affinity for a particular antigen. Some antigen-bindingfragments are composed of portions of intact antibodies that retainantigen-binding specificity of the parent antibody molecule. Forexample, antigen-binding fragments may comprise at least one variableregion (either a heavy chain or light chain variable region) or one ormore CDRs of an antibody known to bind a particular antigen. Examples ofsuitable antigen-binding fragments include, without limitation diabodiesand single-chain molecules as well as Fab, F(ab′)2, Fc, Fabc, and Fvmolecules, single chain (Sc) antibodies, individual antibody lightchains, individual antibody heavy chains, chimeric fusions betweenantibody chains or CDRs and other proteins, protein scaffolds, ormolecules, heavy chain monomers or dimers, light chain monomers ordimers, dimers consisting of one heavy and one light chain, and thelike. All antibody isotypes may be used to produce antigen-bindingfragments. Additionally, antigen-binding fragments may includenon-antibody proteinaceous frameworks that may successfully incorporatepolypeptide segments in an orientation that confers affinity for a givenantigen of interest, such as protein scaffolds. Antigen-bindingfragments may be recombinantly produced or produced by enzymatic orchemical cleavage of intact antibodies. The phrase “an antibody orantigen-binding fragment thereof” may be used to denote that a givenantigen-binding fragment incorporates one or more amino acid segments ofthe antibody referred to in the phrase.

“Antibody compositions” refer to antibodies or binding fragments thereofthat are coupled with at least one pharmaceutically acceptable carrier,chemotherapeutic agent, or diagnostic moiety, such as111In-1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid(¹¹¹In-DOTA).

“Specific binding” refers to the ability of an antibody, orantigen-binding fragment, to bind to a particular biomolecule with anaffinity that is greater than that with which it may bind otherbiomolecules.

The embodiments described herein are not limited to particular methods,reagents, compounds, compositions or biological systems, which can, ofcourse, vary. Furthermore, the terminology used herein is for thepurpose of describing particular antibodies or antigen-binding fragmentsonly, and is not intended to be limiting.

GD2-Specific Antibodies

Described herein are isolated antibodies or antigen-binding fragmentsthat specifically bind the ganglioside GD2. In one embodiment, theantibody or antigen-binding fragment thereof is a monoclonal antibody orantigen-binding fragment; however, other embodiments includeGD2-specific polyclonal antibodies and derivatives or fragments ofantibodies that retain specificity for GD2. The general structure of anantibody molecule comprises an antigen binding domain, which includesheavy and light chains, and the Fc domain, which serves a variety offunctions, including complement fixation. In some embodiments, theantibodies or antigen-binding fragments mediate complement-dependentcytotoxicity.

There are five classes of immunoglobulins wherein the primary structureof the heavy chain, in the Fc region, determines the immunoglobulinclass. Specifically, the alpha, delta, epsilon, gamma, and mu chainscorrespond to IgA, IgD, IgE, IgG and IgM isotypes, respectively. Thedescribed antibodies or antigen-binding fragments include all isotypesand synthetic multimers of the four-chain immunoglobulin structure. Thedescribed antibodies or antigen-binding fragments also include the IgYisotype generally found in hen or turkey serum and hen or turkey eggyolk. Antibodies or antigen-binding fragments non-covalently,specifically, and reversibly bind an antigen.

The antibodies or antigen-binding fragments of the disclosed subjectmatter may be derived from any species. For example, the antibodies orantigen-binding fragments may be mouse, rat, goat, horse, swine, bovine,chicken, rabbit, donkey, human, and the like. For use in the treatmentof humans, non-human derived antibodies or antigen-binding fragments maybe genetically or structurally altered to be less antigenic uponadministration to a human patient.

In some embodiments, the antibodies or antigen-binding fragments arechimeric. As used herein, the term “chimeric” antibody, orantigen-binding fragment, means an antibody, or antigen-binding fragmentthereof, having at least some portion of at least one variable domainderived from the antibody amino acid sequence of a non-human mammal, arodent, or a reptile, while the remaining portions of the antibody, orantigen-binding fragment thereof, are derived from a human. For example,a chimeric antibody may comprise a mouse antigen binding domain with ahuman Fc or other such structural domain.

In some embodiments, the antibodies are humanized antibodies. Humanizedantibodies may be chimeric immunoglobulins, immunoglobulin chains orfragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or otherantigen-binding subsequences of antibodies) that contain minimalsequence derived from non-human immunoglobulin. For the most part,humanized antibodies are human immunoglobulins (recipient antibody) inwhich residues from a complementary-determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity, and capacity. In some instances, Fv frameworkregion (FWR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibodies maycomprise residues which are found neither in the recipient antibody norin the imported CDR or framework sequences. These modifications are madeto further refine and optimize antibody performance. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FWR regions are those of a humanimmunoglobulin sequence. The humanized antibody optimally also willcomprise at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. For further details, see Joneset al., 321 Nature 522-5 (1986); Reichmann et al., 332 Nature 323-9(1988); and Presta, 2 Curr. Op. Struct. Biol. 593-6 (1992).

In some cases, the described antibodies are human antibodies or antigenbinding fragments thereof. As used herein, the term “human antibody”means that the antibody is either solely from human origin or anantibody in which the variable and constant domain sequences are humansequences. The term encompasses antibodies with sequences derived from(i.e., that utilize) human genes, but which have been changed, e.g., todecrease possible immunogenicity, increase affinity, eliminate cysteinesthat may cause undesirable folding, etc. The term encompasses suchantibodies produced recombinantly in non-human cells, which may impartglycosylation not typical of human cells.

The antibodies or antigen-binding fragments described herein may belabeled or otherwise conjugated to various chemical or biomoleculemoieties, for example, for therapeutic or diagnostic applications. Themoieties may be cytotoxic, for example, bacterial toxins, viral toxins,radioisotopes, and the like. The moieties may be detectable labels, forexample, fluorescent labels, radiolabels, biotin, and the like, forexample, ¹¹¹In-DOTA, ¹¹¹In-diethylenetriaminepentaacetic acid (DTPA), orradionuclides, such as, but not limited to, lead-212, bismuth-212,astatine-211, iodine-131, scandium-47, rhenium-186, rhenium-188,yttrium-90, iodine-123, iodine-124, iodine-125, bromine-77, indium-111,and fissionable nuclides such as boron-10 or an actinide.

In some embodiments, the antibodies or antigen-binding fragments areconjugated to one or more chemotherapeutic agents such as, but notlimited to radionuclides, toxins, and cytotoxic and cytostatic agents.In other embodiments the antibodies or antigen-binding fragments areused in combination with one or more chemotherapeutic agents. Theantibodies or antigen-binding fragments described herein may be usedalone or with (e.g., coadministered or conjugated to) a biomolecule orchemotherapeutic agent such as a cytotoxic or cytostatic agent. In someembodiments, the chemotherapeutic agent is a radionuclide, including,but not limited to lead-212, bismuth-212, astatine-211, iodine-131,scandium-47, rhenium-186, rhenium-188, yttrium-90, iodine-123,iodine-124, iodine-125, bromine-77, indium-111, and fissionable nuclidessuch as boron-10 or an actinide. In other embodiments, thechemotherapeutic agent is a toxin or cytotoxic drug, pokeweed antiviralprotein, abrin, ricin and each of their A chains, momordin, saporin,bryodin 1, bouganin, gelonin, Diphtheria toxin, Pseudomonas exotoxin,Shiga toxin, calicheamicin, maytansinoid, altretamine, actinomycin D,plicamycin, puromycin, gramicidin D, doxorubicin, colchicine,cytochalasin B, cyclophosphamide, emetine, maytansine, amsacrine,cisplastin, etoposide, etoposide orthoquinone, teniposide, daunorubicin,gemcitabine, doxorubicin, mitoxantraone, bisanthrene, Bleomycin,methotrexate, pemetrexed, cisplatinum, vindesine, adriamycin,vincristine, vinblastine, BCNU, a taxane (e.g., Taxol™), tarceva,avastin, mitomycin, 5-fluorouracil, cyclophosphamide, certain cytokinessuch as TNF-alpha and TNF-beta, and the like. Methods of conjugation ofantibodies or antigen-binding fragments to such agents are known in theliterature.

Antibody specificity is primarily determined by the six CDR regions,especially H chain CDR3 (Kala et al., 132 J. Biochem. 535-41 (2002);Morea et al., 275 J. Mol. Biol. 269-94 (1998); and, Chothia et al., 196J. Mol. Biol. 901-17 (1987)). Antibody framework regions, however, canplay a role in antigen-antibody interactions (Panka et al., 85 Proc.Natl. Acad. Sci. USA 3080-4 (1988)), particularly in influencing theconformation of CDR loops (Foote et al., 224 J. Mol. Biol. 487-99(1992)). Thus, the described antibodies or antigen-binding fragments maycomprise any combination of H or L chain CDR or FWR regions that conferspecificity to GD2. Domain shuffling experiments, which are routinelycarried out in the art (Jirholt et al., 215 Gene 471-6 (1998); Söderlindet al., 18 Nature Biotechnology 852-6 (2000)), may be employed togenerate antibodies that specifically bind GD2 according to thespecifications described and exemplified herein. Antibodies orantigen-binding fragments generated by such domain shuffling experimentsare within the scope of the antibodies or antigen-binding fragmentsdescribed herein. Furthermore, CDRs may also be arranged to bind a givenantigen by engineering antibody-like proteins to serve as CDRscaffolding (Nicaise et al., 13 Protein Sci. 1882 (2004)). Suchantigen-binding proteins are within the scope of the antibodiesdescribed herein.

The antibodies or antigen-binding fragments described herein can occurin a variety of forms, but will include one or more of the antibodysegments shown in Table 1.

TABLE 1 Antibody segments of the described antibodies andantigen-binding fragments thereof (“Lc” denotes light chain and “Hc”denotes heavy chain). Amino Acid DNA AB527 Antibody Segment SEQ ID NO.SEQ ID NO. Lc CDR1 26 18 Lc CDR2 27 19 Lc CDR3 28 20 Lc FWR1 29 21 LcFWR2 30 22 Lc FWR3 31 23 Lc variable domain 32 24 Light chain 42 41 HcCDR1 10 2 Hc CDR2 11 3 Hc CDR3 12 4 Hc FWR1 13 5 Hc FWR2 14 6 Hc FWR3 157 Hc variable domain 16 8 Heavy chain 40 39

In some embodiments, the antibodies or antigen-binding fragments mayinclude a heavy chain CDR1 amino acid sequence substantially the sameas, or identical to, SEQ ID NO: 10. In some embodiments, the antibodiesor antigen-binding fragments may include a heavy chain CDR2 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 11. Insome embodiments, antibodies or antigen-binding fragments may include aheavy chain CDR3 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 12. In some embodiments, antibodies orantigen-binding fragments may include a light chain CDR1 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 26. Insome embodiments, antibodies or antigen-binding fragments may include alight chain CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 27. In some embodiments, antibodies orantigen-binding fragments may include a light chain CDR3 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 28. Insome embodiments, antibodies or antigen-binding fragments may include aheavy chain CDR1 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 10; a CDR2 amino acid sequence substantiallythe same as, or identical to, SEQ ID NO: 11; and a CDR3 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 12. Insome embodiments, antibodies or antigen-binding fragments may include alight chain CDR1 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 26; a CDR2 amino acid sequence substantiallythe same as, or identical to, SEQ ID NO: 27; and a CDR3 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 28. Insome embodiments, antibodies or antigen-binding fragments may include aheavy chain and a light chain, wherein the heavy chain has a CDR1 aminoacid sequence substantially the same as, or identical to, SEQ ID NO: 10;a CDR2 amino acid sequence substantially the same as, or identical to,SEQ ID NO: 11; and a CDR3 amino acid sequence substantially the same as,or identical to, SEQ ID NO: 12; and the light chain has a CDR1 aminoacid sequence substantially the same as, or identical to, SEQ ID NO: 26;a CDR2 amino acid sequence substantially the same as, or identical to,SEQ ID NO: 27; and a CDR3 amino acid sequence substantially the same as,or identical to, SEQ ID NO: 28. Antigen-binding arrangements of CDRs mayalso be engineered using antibody-like proteins as CDR scaffolding. Suchengineered antigen-binding proteins are within the scope of thedisclosure.

In some embodiments, antibodies or antigen-binding fragments may includea heavy chain FWR1 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 13. In some embodiments, antibodies orantigen-binding fragments may include a heavy chain FWR2 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 14. Insome embodiments, antibodies or antigen-binding fragments may include aheavy chain FWR3 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 15. In some embodiments, antibodies orantigen-binding fragments may include a light chain FWR1 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 29. Insome embodiments, antibodies or antigen-binding fragments may include alight chain FWR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 30. In some embodiments, antibodies orantigen-binding fragments may include a light chain FWR3 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 31. Insome embodiments, antibodies or antigen-binding fragments may include aheavy chain having a FWR1 amino acid sequence that is substantially thesame as, or identical to, SEQ ID NO: 13; a FWR2 amino acid sequence thatis substantially the same as, or identical to, SEQ ID NO: 14; and a FWR3amino acid sequence that is substantially the same as, or identical to,SEQ ID NO: 15. In some embodiments, antibodies or antigen-bindingfragments may include a light chain having a FWR1 amino acid sequencethat is substantially the same as, or identical to, SEQ ID NO: 29; aFWR2 amino acid sequence that is substantially the same as, or identicalto, SEQ ID NO: 30; and a FWR3 amino acid sequence that is substantiallythe same as, or identical to, SEQ ID NO: 31. In some embodiments,antibodies or antigen-binding fragments may include a heavy chain and alight chain, wherein the heavy chain includes a FWR1 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 13; a FWR2 aminoacid sequence substantially the same as, or identical to, SEQ ID NO: 14;and a FWR3 amino acid sequence substantially the same as, or identicalto, SEQ ID NO: 15; and the light chain includes a FWR1 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 29; aFWR2 amino acid sequence substantially the same as, or identical to, SEQID NO: 30; and a FWR3 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 31.

In some embodiments, antibodies or antigen-binding fragments may includea heavy chain having a CDR1 amino acid sequence substantially the sameas, or identical to, SEQ ID NO: 10; a CDR2 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 11; and a CDR3amino acid sequence substantially the same as, or identical to, SEQ IDNO: 12; a FWR1 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 13; a FWR2 amino acid sequence substantiallythe same as, or identical to, SEQ ID NO: 14; and a FWR3 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 15. Insome embodiments, the antibodies or antigen-binding fragments include alight chain having a CDR1 amino acid sequence substantially the same as,or identical to, SEQ ID NO: 26; a CDR2 amino acid sequence substantiallythe same as, or identical to, SEQ ID NO: 27; and a CDR3 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 28; aFWR1 amino acid sequence substantially the same as, or identical to, SEQID NO: 29; a FWR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 30; and a FWR3 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 31. In someembodiments, the antibodies or antigen-binding fragments include a heavyand a light chain, wherein the heavy chain includes a CDR1 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 10; aCDR2 amino acid sequence substantially the same as, or identical to, SEQID NO: 11; and a CDR3 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 12; a FWR1 amino acid sequence substantiallythe same as, or identical to, SEQ ID NO: 13; a FWR2 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 14; and a FWR3amino acid sequence substantially the same as, or identical to, SEQ IDNO: 15; and the light chain includes a CDR1 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 26; a CDR2 aminoacid sequence substantially the same as, or identical to, SEQ ID NO: 27;and a CDR3 amino acid sequence substantially the same as, or identicalto, SEQ ID NO: 28; a FWR1 amino acid sequence substantially the same as,or identical to, SEQ ID NO: 29; a FWR2 amino acid sequence substantiallythe same as, or identical to, SEQ ID NO: 30; and a FWR3 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 31.Antigen-binding arrangements of CDRs and FWRs may also be engineeredusing antibody-like proteins as CDR scaffolding. Such engineeredantigen-binding proteins are within the scope of the disclosure.

In some embodiments, the antibodies or antigen-binding fragmentsdescribed herein have a heavy chain that includes the amino acidsequence substantially the same as, or identical to, SEQ ID NO: 40. Insome embodiments, the antibodies or antigen-binding fragments describedherein have a light chain that includes the amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 42. The describedantibodies or antigen-binding fragments may have a heavy and a lightchain, wherein the heavy chain includes the amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 40 and the lightchain includes the amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 42.

Also described are polynucleotides that encode antibodies orantigen-binding fragments that specifically bind to GD2. In someembodiments, the polynucleotides encode an antibody or antigen-bindingfragment thereof having a heavy chain CDR1 sequence substantially thesame as, or identical to, SEQ ID NO: 10, for example SEQ ID NO: 2. Insome embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain CDR2 substantiallythe same as, or identical to, SEQ ID NO: 11, for example SEQ ID NO: 3.In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain CDR3 substantiallythe same as, or identical to, SEQ ID NO: 12, for example SEQ ID NO: 4.In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a light chain CDR1 substantiallythe same as, or identical to, SEQ ID NO: 26, for example SEQ ID NO: 18.In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a light chain CDR2 substantiallythe same as, or identical to, SEQ ID NO: 27, for example SEQ ID NO: 19.In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a light chain CDR3 substantiallythe same as, or identical to, SEQ ID NO: 28, for example SEQ ID NO: 20.The polynucleotides may encode an antibody or antigen-binding fragmentthereof having a heavy chain with a CDR1 substantially the same as, oridentical to, SEQ ID NO: 10, for example SEQ ID NO: 2; a CDR2substantially the same as, or identical to, SEQ ID NO: 11, for exampleSEQ ID NO: 3; and a CDR3 substantially the same as, or identical to, SEQID NO: 12, for example SEQ ID NO: 4. The polynucleotides may encode anantibody or antigen-binding fragment thereof having a light chain CDR1substantially the same as, or identical to, SEQ ID NO: 26, for exampleSEQ ID NO: 18; a CDR2 substantially the same as, or identical to, SEQ IDNO: 27, for example SEQ ID NO: 19; and a CDR3 substantially the same as,or identical to, SEQ ID NO: 28, for example SEQ ID NO: 20. Thepolynucleotides may encode an antibody or antigen-binding fragmentthereof having a heavy chain CDR1 substantially the same as, oridentical to, SEQ ID NO: 10, for example SEQ ID NO: 2; a CDR2 encoded bya nucleotide sequence substantially the same as, or identical to, SEQ IDNO: 11, for example SEQ ID NO: 3; and a CDR3 encoded by a nucleotidesequence substantially the same as, or identical to, SEQ ID NO: 12, forexample SEQ ID NO: 4; and a light chain CDR1 substantially the same as,or identical to, SEQ ID NO: 26, for example SEQ ID NO: 18; a CDR2substantially the same as, or identical to, SEQ ID NO: 27, for exampleSEQ ID NO: 19; and a CDR3 substantially the same as, or identical to,SEQ ID NO: 28, for example SEQ ID NO: 20.

In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain FWR1 substantiallythe same as, or identical to, SEQ ID NO: 13, for example SEQ ID NO: 5.In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain FWR2 substantiallythe same as, or identical to, SEQ ID NO: 14, for example SEQ ID NO: 6.In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain FWR3 substantiallythe same as, or identical to, SEQ ID NO: 15, for example SEQ ID NO: 7.In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a light chain FWR1 substantiallythe same as, or identical to, SEQ ID NO: 29, for example SEQ ID NO: 21.In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a light chain FWR2 substantiallythe same as, or identical to, SEQ ID NO: 30, for example SEQ ID NO: 22.In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a light chain FWR3 substantiallythe same as, or identical to, SEQ ID NO: 31, for example SEQ ID NO: 23.In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain FWR1 substantiallythe same as, or identical to, SEQ ID NO: 13, for example SEQ ID NO: 5; aFWR2 substantially the same as, or identical to, SEQ ID NO: 14, forexample SEQ ID NO: 6; and a FWR3 substantially the same as, or identicalto, SEQ ID NO: 15, for example SEQ ID NO: 7. In some embodiments, thepolynucleotides encode an antibody or antigen-binding fragment thereofhaving a light FWR1 substantially the same as, or identical to, SEQ IDNO: 29, for example SEQ ID NO: 21; a FWR2 substantially the same as, oridentical to, SEQ ID NO: 30, for example SEQ ID NO: 22; and a FWR3substantially the same as, or identical to, SEQ ID NO: 31, for exampleSEQ ID NO: 23. In some embodiments, the polynucleotides encode anantibody or antigen-binding fragment thereof having a heavy chain and alight chain, wherein a heavy chain FWR1 is substantially the same as, oridentical to, SEQ ID NO: 13, for example SEQ ID NO: 5; a heavy chainFWR2 is substantially the same as, or identical to, SEQ ID NO: 14, forexample SEQ ID NO: 6; and a heavy chain FWR3 is substantially the sameas, or identical to, SEQ ID NO: 15, for example SEQ ID NO: 7; and alight chain FWR1 is substantially the same as, or identical to, SEQ IDNO: 29, for example SEQ ID NO: 21; a light chain FWR2 is substantiallythe same as, or identical to, SEQ ID NO: 30, for example SEQ ID NO: 22;and a light chain FWR3 substantially the same as, or identical to, SEQID NO: 31, for example SEQ ID NO: 23.

In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain CDR1 substantiallythe same as, or identical to, SEQ ID NO: 10, for example SEQ ID NO: 2; aheavy chain CDR2 substantially the same as, or identical to, SEQ ID NO:11, for example SEQ ID NO: 3; and a heavy chain CDR3 substantially thesame as, or identical to, SEQ ID NO: 12, for example SEQ ID NO: 4; aheavy chain FWR1 substantially the same as, or identical to, SEQ ID NO:13, for example SEQ ID NO: 5; a heavy chain FWR2 substantially the sameas, or identical to, SEQ ID NO: 14, for example SEQ ID NO: 6; and aheavy chain FWR3 substantially the same as, or identical to, SEQ ID NO:15, for example SEQ ID NO: 7. In some embodiments, the polynucleotidesencode an antibody or antigen-binding fragment thereof having a lightchain CDR1 substantially the same as, or identical to, SEQ ID NO: 26,for example SEQ ID NO: 18; a light chain CDR2 substantially the same as,or identical to, SEQ ID NO: 27, for example SEQ ID NO: 19; and a lightchain CDR3 substantially the same as, or identical to, SEQ ID NO: 28,for example SEQ ID NO: 20; a light chain FWR1 substantially the same as,or identical to, SEQ ID NO: 29, for example SEQ ID NO: 21; a light chainFWR2 substantially the same as, or identical to, SEQ ID NO: 30, forexample SEQ ID NO: 22; and a light chain FWR3 substantially the same as,or identical to, SEQ ID NO: 31, for example SEQ ID NO: 23.

In some embodiments, the polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy and a light chain,wherein the polynucleotides encode a heavy chain CDR1 substantially thesame as, or identical to, SEQ ID NO: 10, for example SEQ ID NO: 2; aheavy chain CDR2 substantially the same as, or identical to, SEQ ID NO:11, for example SEQ ID NO: 3; a heavy chain CDR3 substantially the sameas, or identical to, SEQ ID NO: 12, for example SEQ ID NO: 4; a heavychain FWR1 substantially the same as, or identical to, SEQ ID NO: 13,for example SEQ ID NO: 5; a heavy chain FWR2 substantially the same as,or identical to, SEQ ID NO: 14, for example SEQ ID NO: 6; and a heavychain FWR3 substantially the same as, or identical to, SEQ ID NO: 15,for example SEQ ID NO: 7; and a light chain CDR1 substantially the sameas, or identical to, SEQ ID NO: 26, for example SEQ ID NO: 18; a lightchain CDR2 substantially the same as, or identical to, SEQ ID NO: 27,for example SEQ ID NO: 19; a light chain CDR3 substantially the same as,or identical to, SEQ ID NO: 28, for example SEQ ID NO: 20; a light chainFWR1 substantially the same as, or identical to, SEQ ID NO: 29, forexample SEQ ID NO: 21; a light chain FWR2 substantially the same as, oridentical to, SEQ ID NO: 30, for example SEQ ID NO: 22; and a lightchain FWR3 substantially the same as, or identical to, SEQ ID NO: 31,for example SEQ ID NO: 23.

Polynucleotides encoding engineered antigen-binding proteins also arewithin the scope of the disclosure.

In some embodiments, the polynucleotides described (and the peptidesthey encode) include a leader sequence. Any leader sequence known in theart may be employed. The leader sequence may include, but is not limitedto, a restriction site or a translation start site. In some embodiments,the leader sequence has the nucleic acid sequenceATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTACACAGC (SEQ ID NO:43). In some embodiments, the leader sequence encodes the amino acidsequence MGWSCIILFLVATATGVHS (SEQ ID NO: 44).

Because of the natural sequence variation likely to exist among heavyand light chains and the genes encoding them, one would expect to findsome level of variation within the amino acid sequences or the genesencoding the antibodies or antigen-binding fragments described herein,with little or no impact on their unique binding properties (e.g.,specificity and affinity). Such an expectation is due in part to thedegeneracy of the genetic code, as well as to the evolutionary successof conservative amino acid sequence variations, which do not appreciablyalter the nature of the encoded protein. Accordingly, some embodimentsinclude antibodies or antigen-binding fragments having 90%, 95%, 96%,97%, 98%, or 99% homology to the antibodies or antigen-binding fragmentsherein. Other embodiments include GD-2 specific antibodies orantigen-binding fragments, that have framework, scaffold, or othernon-binding regions that do not share significant homology with theantibodies and antigen-binding fragments described herein, but doincorporate one or more CDRs or other sequences needed to confer bindingthat are 90%, 95%, 96%, 97%, 98%, or 99% homologous to such sequencesdescribed herein.

The antibodies or antigen-binding fragments described herein includevariants having single or multiple amino acid substitutions, deletions,or additions that retain the biological properties (e.g., bindingaffinity or immune effector activity) of the described antibodies orantigen-binding fragments. The skilled person may produce variantshaving single or multiple amino acid substitutions, deletions, oradditions. These variants may include: (a) variants in which one or moreamino acid residues are substituted with conservative or nonconservativeamino acids, (b) variants in which one or more amino acids are added toor deleted from the polypeptide, (c) variants in which one or more aminoacids include a substituent group, and (d) variants in which thepolypeptide is fused with another peptide or polypeptide such as afusion partner, a protein tag or other chemical moiety, that may conferuseful properties to the polypeptide, such as, for example, an epitopefor an antibody, a polyhistidine sequence, a biotin moiety and the like.Antibodies or antigen-binding fragments described herein may includevariants in which amino acid residues from one species are substitutedfor the corresponding residue in another species, either at theconserved or nonconserved positions. In other embodiments, amino acidresidues at nonconserved positions are substituted with conservative ornonconservative residues. The techniques for obtaining these variants,including genetic (suppressions, deletions, mutations, etc.), chemical,and enzymatic techniques, are known to the person having ordinary skillin the art.

The antibodies or antigen-binding fragments described herein may embodyseveral antibody isotypes, such as IgM, IgD, IgG, IgA and IgE. Antibodyor antigen-binding fragment thereof specificity is largely determined bythe amino acid sequence, and arrangement, of the CDRs. Therefore, theCDRs of one isotype may be transferred to another isotype withoutaltering antigen specificity. Alternatively, techniques have beenestablished to cause hybridomas to switch from producing one antibodyisotype to another (isotype switching) without altering antigenspecificity. Accordingly, such antibody isotypes are within the scope ofthe described antibodies or antigen-binding fragments.

Altering the isotype of an antibody may be useful for combiningdifferent effector functions, conferred by the antibody constant region,with antigen specificity. For example, IgG is produced in monomericform, is effective at activating complement, and is commonly found inthe plasma and intracellular compartments of the body. IgA is producedas a monomer and a J-chain-associated dimer. IgA is most commonly foundin the luminal spaces of the body and breast milk, because the dimericform may be transported across epithelial cell barriers. Like IgA, IgMcan also form polymers, however, it tends to form pentamers, whenassociated with J-chain, and pentamers and hexamers not associated withJ-chain. These highly polymeric antibodies serve to increase overallantigen-binding capability by increasing avidity without reducingaffinity. Both polymeric forms of IgM can activate complementefficiently, which can result in complement-dependent cytotoxicity(CDC).

The antibodies or antigen-binding fragments described herein, in someembodiments, are polymeric IgM antibodies or antigen-binding fragments.In some embodiments, the antibodies or antigen-binding fragments arepentameric IgM antibodies or antigen-binding fragments that areassociated with J-chain. In other embodiments, the antibodies orantigen-binding fragments are hexameric IgM antibodies orantigen-binding fragments that are not associated with J-chain. In someembodiments, the antibodies or antigen-binding fragments are pentamericIgM antibodies or antigen-binding fragments that are not associated withJ-chain. In some embodiments, the antibodies or antigen-bindingfragments are hexameric IgM antibodies or antigen-binding fragments thatare associated with J-chain.

The antibodies or antigen-binding fragments described herein havebinding affinities (in M) for GD2 that include a dissociation constant(K_(D)) of less than 1×10⁻². In some embodiments, the K_(D) is less than1×10⁻³. In other embodiments, the K_(D) is less than 1×10⁻⁴. In someembodiments, the K_(D) is less than 1×10⁻⁵. In still other embodiments,the K_(D) is less than 1×10⁻⁶, 2×10⁻⁶, 3×10⁻⁶, 4×10⁻⁶, 5×10⁻⁶, 6×10⁻⁶,7×10⁻⁶, 8×10⁻⁶, or 9×10⁻⁶. In other embodiments, the K_(D) is less than1×10⁻⁷, 2×10⁻⁷, or 3×10⁻⁷, 2×10⁻⁷, 3×10⁻⁷, 4×10⁻⁷, 5×10⁻⁷, 6×10⁻⁷,7×10⁻⁷, 8×10⁻⁷, or 9×10⁻⁷. In other embodiments, the K_(D) is less than1×10⁻⁸, 2×10⁻⁸, 3×10⁻⁸, 4×10⁻⁸, 5×10⁻⁸, 6×10⁻⁸, 7×10⁻⁸, 8×10⁻⁸, or9×10⁻⁸. In other embodiments, the K_(D) is less than 1×10⁻⁹, 2×10⁻⁹,3×10⁻⁹, 4×10⁻⁹, 5×10⁻⁹, 6×10⁻⁹, 7×10⁻⁹, 8×10⁻⁹, or 9×10⁻⁹. In otherembodiments, the K_(D) is less than 1×10⁻¹⁰, 2×10⁻¹⁰, 3×10⁻¹⁰, 2×10⁻¹⁰,3×10⁻¹⁰, 4×10⁻¹⁰, 5×10⁻¹⁰, 6×10⁻¹⁰, 7×10⁻¹⁰, 8×10⁻¹⁰, or 9×10⁻¹⁰. Instill other embodiments, the K_(D) is less than 1×10⁻¹¹, 2×10⁻¹¹,3×10⁻¹¹, 4×10⁻¹¹, 5×10⁻¹¹, 6×10⁻¹¹, 7×10⁻¹¹, 8×10⁻¹¹, or 9×10⁻¹¹. Insome embodiments, the K_(D) is less than 1×10⁻¹². In other embodiments,the K_(D) is less than 1×10⁻¹³. In other embodiments, the K_(D) is lessthan 1×10⁻¹⁴. In still other embodiments, the K_(D) is less than1×10⁻¹⁵. In preferred embodiments, the K_(D) is 4.5×10⁻⁹ or less.

The antibodies or antigen-binding fragments described herein, in someembodiments, have specific binding affinities for GD2 in contrast witheach of GD1a, GM2 or GM3. In some embodiments, the K_(D) for GD2 differsfrom the K_(D) for each of GD1a, GM2 or GM3 by at least 3-fold,preferably 10-fold.

The antibodies or antigen-binding fragments described herein may bemodified, e.g., by the covalent attachment of any type of molecule tothe antibody or antigen-binding fragment thereof such that covalentattachment does not prevent the antibody or antigen-binding fragmentthereof from binding to its epitope. Examples of suitable modificationsinclude, but are not limited to glycosylation, acetylation, pegylation,phosphorylation, amidation, and the like. In some embodiments theantibodies or antigen-binding fragments may themselves be derivatized byknown protecting/blocking groups, proteolytic cleavage, linkage to acellular ligand or other proteins, and the like. The antibodies orantigen-binding fragments may have post-translational moieties thatimprove upon antibody or antigen-binding fragment thereof activity orstability. These moieties include sulfur, methyl, carbohydrate,phosphorus as well as other chemical groups commonly found onimmunoglobulin molecules. Furthermore, the antibodies or antigen-bindingfragments may contain one or more non-classical amino acids.

Antibodies or antigen-binding fragments described herein may be labeledwith or conjugated to toxic or non-toxic moieties. Toxic moietiesinclude, for example, bacterial toxins, viral toxins, plant toxins,fungal toxins, radioisotopes, and the like. Antibodies orantigen-binding fragments may be labeled for use in biological assays(e.g., radioisotope labels, fluorescent labels) to aid in detection ofthe antibody or antigen-binding fragment. Antibodies or antigen-bindingfragments may also be labeled or conjugated for diagnostic ortherapeutic purposes, e.g., with radioactive isotopes that deliverradiation directly to a desired site for applications such asradioimmunotherapy (Garmestani et al., 28 Nucl. Med. Biol. 409 (2001)),imaging techniques and radioimmunoguided surgery or labels that allowfor in vivo imaging or detection of specific antibody/antigen complexes.Antibodies or antigen-binding fragments may also be conjugated withtoxins to provide an immunotoxin (see, Kreitman, R. J., 31 Adv. DrugDel. Rev. 53 (1998)).

Described herein are compositions comprising at least one describedantibody or antigen-binding fragment thereof and a pharmaceuticallyacceptable carrier. Such compositions are useful, for example, foradministration to patients to treat cancer, such as those described andexemplified herein. The compositions may be formulated as any of variouspreparations that are known and suitable in the art, including thosedescribed and exemplified herein. In some embodiments, the compositionsare aqueous formulations. Aqueous solutions may be prepared by admixingthe antibodies or antigen-binding fragments in water or suitablephysiologic buffer, and optionally adding suitable colorants, flavors,preservatives, stabilizing and thickening agents and the like asdesired. Aqueous suspensions may also be made by dispersing theantibodies or antigen-binding fragments in water or physiologic bufferwith viscous material, such as natural or synthetic gums, resins,methylcellulose, sodium carboxymethylcellulose, and other well-knownsuspending agents.

Also included are liquid formulations and solid form preparations whichare intended to be converted, shortly before use, to liquidpreparations. Such liquids include solutions, suspensions, syrups,slurries, and emulsions. Liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats or oils); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, orfractionated vegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). These preparations maycontain, in addition to the active agent, colorants, flavors,stabilizers, buffers, artificial and natural sweeteners, dispersants,thickeners, solubilizing agents, and the like. The compositions may bein powder or lyophilized form for constitution with a suitable vehiclesuch as sterile water, physiological buffer, saline solution, oralcohol, before use.

The compositions may be formulated for injection into a subject. Forinjection, the compositions described may be formulated in aqueoussolutions such as water or alcohol, or in physiologically compatiblebuffers such as Hanks's solution, Ringer's solution, or physiologicalsaline buffer. The solution may contain one or more formulatory agentssuch as suspending, stabilizing or dispersing agents. Injectionformulations may also be prepared as solid form preparations which areintended to be converted, shortly before use, to liquid formpreparations suitable for injection, for example, by constitution with asuitable vehicle, such as sterile water, saline solution, or alcohol,before use.

The compositions may be formulated in sustained release vehicles ordepot preparations. Such long-acting formulations may be administered byimplantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the compositions may beformulated with suitable polymeric or hydrophobic materials (forexample, as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt. Liposomes and emulsions are well-known examples of deliveryvehicles suitable for use as carriers for hydrophobic drugs.

Disclosed herein are methods for detecting GD-2-expressing cells usingthe described antibodies, or antigen-binding fragments, either in vivoor in vitro. Some embodiments make use of disclosed antibodies, orantigen-binding fragments, that are conjugated to detectable labels suchas fluorescent labels, radiolabels, biotin, enzymes and the like, forexample ¹¹¹In-DOTA, ¹¹¹In-DTPA, or radionuclides, including, but notlimited to, lead-212, bismuth-212, astatine-211, iodine-131,scandium-47, rhenium-186, rhenium-188, yttrium-90, iodine-123,iodine-124, iodine-125, bromine-77, indium-111, and fissionable nuclidessuch as boron-10 or an actinide. For example, a detectably labeledantibody, or antigen-binding fragment, may be administered to a subjectto detect and localize cells in the subject that express GD2. Suchmethods could also be used to detect cells or tissues where GD2 ishighly expressed relative to other cells or tissues in the subject.Alternatively, one embodiment of the disclosed method for detectingGD-2-expressing cells may include using a detectably labeled antibody orantigen-binding fragment, described herein, to detect or quantify theexpression of GD2 by cells obtained from a subject, such as cellsobtained from a blood sample or tissue biopsy.

Alternatively, the described methods for detecting GD-2-expressing cellsusing the described antibodies, or antigen-binding fragments, may beperformed using GD2-specific antibodies that are not labeled. Forexample, in one embodiment, GD2-expressing cells may be detected in asubject by first administering to the subject a GD2-specific antibody,or antigen-binding fragment, described herein followed by administrationof a detectably labeled secondary antibody capable of binding theGD2-specific antibody, or antigen-binding fragment, administeredinitially. A similar methodology could be used to detect GD2-expressingcells in vitro.

Also described herein are methods for treating or preventing diseases insubjects in need of such treatment or prevention. In some aspects, themethods can include identifying a subject in need of treatment orprevention of GD2-associated diseases, such as cancer, e.g., melanoma.Other embodiments include an antibody or antigen-binding fragmentthereof that is labeled or conjugated for diagnostic purposes, e.g.,with radioactive isotopes that deliver radiation directly to a desiredsite, such as imaging techniques or detection of specificantibody/antigen complexes. In one embodiment, the methods compriseadministering to the subject a GD2-specific antibody or antigen-bindingfragment, such as a recombinant, human, GD2-specific IgM antibody in anamount effective to treat or prevent disease. In one aspect the methodsinclude administering to the subject a composition, such as thosedescribed and exemplified herein, the composition comprising apharmaceutically acceptable carrier and at least one antibody orantigen-binding fragment thereof that specifically binds to GD2, in anamount effective to treat or prevent disease. In one embodiment, themethods comprise administering to the subject at least one antibody orantigen-binding fragment, such as the antibodies or antigen-bindingfragments described and exemplified herein, that specifically binds toGD2, in an amount effective to treat or prevent disease. In oneembodiment, the methods comprise administering to the subject at leastone GD2-specific antibody or antigen-binding fragment thereof labeled orconjugated to toxic or non-toxic moieties exemplified herein.

The antibodies or antigen-binding fragments described herein may beadministered orally in any acceptable dosage form such as capsules,tablets, aqueous suspensions, solutions or the like. The antibodies orantigen-binding fragments may also be administered parenterallyincluding but not limited to: subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intranasal, topically,intrathecal, intrahepatic, intralesional, and intracranial injection orinfusion techniques. Alternatively, the antibodies or antigen-bindingfragments will be intravenously or intraperitoneally, for example, byinjection.

The subject may be any animal, and preferably is a mammal such as amouse, rat, hamster, guinea pig, rabbit, cat, dog, monkey, donkey, cow,horse, pig, and the like. Most preferably, the mammal is a human. Insome embodiments, subjects may be administered at least one anti-GD2antibody or antigen-binding fragment thereof in a daily dose range of0.01 μg to 500 mg of antibody or antigen-binding fragment thereof per kgof the weight of the subject. The dose administered to the subject mayalso be measured in terms of total amount of the at least one anti-GD2antibody or antigen-binding fragment thereof administered per day. Insome embodiments, a subject is administered 5 to 5000 milligrams of atleast one anti-GD2 antibody or antigen-binding fragment thereof per day.In some embodiments, a subject is administered up to 10 milligrams of atleast one anti-GD2 antibody or antigen-binding fragment thereof per day.In some embodiments, a subject is administered up to 100 milligrams ofat least one anti-GD2 antibody or antigen-binding fragment thereof perday. In some embodiments, a subject is administered up to 250 milligramsof at least one anti-GD2 antibody or antigen-binding fragment thereofper day. In some embodiments, a subject is administered up to 500milligrams of at least one anti-GD2 antibody or antigen-binding fragmentthereof per day. In some embodiments, a subject is administered up to750 milligrams of at least one anti-GD2 antibody or antigen-bindingfragment thereof per day. In some embodiments, a subject is administeredup to 1000 milligrams of at least one anti-GD2 antibody orantigen-binding fragment thereof per day. In some embodiments, a subjectis administered up to 1500 milligrams of at least one anti-GD2 antibodyor antigen-binding fragment thereof per day. In some embodiments, asubject is administered up to 2000 milligrams of at least one anti-GD2antibody or antigen-binding fragment thereof per day. In someembodiments, a subject is administered up to 2500 milligrams of at leastone anti-GD2 antibody or antigen-binding fragment thereof per day. Insome embodiments, a subject is administered up to 3000 milligrams of atleast one anti-GD2 antibody or antigen-binding fragment thereof per day.In some embodiments, a subject is administered up to 3500 milligrams ofat least one anti-GD2 antibody or antigen-binding fragment thereof perday. In some embodiments, a subject is administered up to 4000milligrams of at least one anti-GD2 antibody or antigen-binding fragmentthereof per day. In some embodiments, a subject is administered up to4500 milligrams of at least one anti-GD2 antibody or antigen-bindingfragment thereof per day. In some embodiments, a subject is administeredup to 5000 milligrams of at least one anti-GD2 antibody orantigen-binding fragment thereof per day. In some embodiments, antibodyor antigen-binding fragment thereof is administered to a subject weeklyor bi-weekly.

Treatment may be initiated with smaller dosages that are less than theoptimum dose of the at least one anti-GD2 antibody or antigen-bindingfragment, followed by an increase in dosage over the course of thetreatment until the optimum effect under the circumstances is reached.If indicated, the total daily dosage may be divided and administered inportions throughout the day.

For effective treatment of GD2-associated diseases, one skilled in theart may recommend a dosage schedule and dosage amount adequate for thesubject being treated. Dosing may occur one to four or more times dailyfor as long as needed. The dosing may occur less frequently if thecompositions are formulated in sustained delivery vehicles. The dosageschedule may also vary depending on the active drug concentration, whichmay depend on the needs of the subject.

Also provided are vectors comprising the polynucleotides describedherein. The vectors can be expression vectors. Recombinant expressionvectors containing a sequence encoding a polypeptide of interest arethus provided. The expression vector may contain one or more additionalsequences such as but not limited to regulatory sequences (e.g.,promoter, enhancer), a selection marker, and a polyadenylation signal.Vectors for transforming a wide variety of host cells are well known andinclude, but are not limited to, plasmids, phagemids, cosmids,baculoviruses, bacmids, bacterial artificial chromosomes (BACs), yeastartificial chromosomes (YACs), as well as other bacterial, yeast andviral vectors.

Recombinant expression vectors within the scope of the descriptioninclude synthetic, genomic, or cDNA-derived nucleic acid fragments thatencode at least one recombinant protein which may be operably linked tosuitable regulatory elements. Such regulatory elements may include atranscriptional promoter, sequences encoding suitable mRNA ribosomalbinding sites, and sequences that control the termination oftranscription and translation. Expression vectors, especially mammalianexpression vectors, may also include one or more nontranscribed elementssuch as an origin of replication, a suitable promoter and enhancerlinked to the gene to be expressed, other 5′ or 3′ flankingnontranscribed sequences, 5′ or 3′ nontranslated sequences (such asnecessary ribosome binding sites), a polyadenylation site, splice donorand acceptor sites, or transcriptional termination sequences. An originof replication that confers the ability to replicate in a host may alsobe incorporated.

The transcriptional and translational control sequences in expressionvectors to be used in transforming vertebrate cells may be provided byviral sources. Exemplary vectors may be constructed as described byOkayama and Berg, 3 Mol. Cell. Biol. 280 (1983).

In some embodiments, the antibody or antigen-binding fragment-codingsequence is placed under control of a powerful constitutive promoter,such as the promoters for the following genes: hypoxanthinephosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase,beta-actin, human myosin, human hemoglobin, human muscle creatine, andothers. In addition, many viral promoters function constitutively ineukaryotic cells and are suitable for use with the describedembodiments. Such viral promoters include without limitation,Cytomegalovirus (CMV) immediate early promoter, the early and latepromoters of SV40, the Mouse Mammary Tumor Virus (MMTV) promoter, thelong terminal repeats (LTRs) of Maloney leukemia virus, HumanImmunodeficiency Virus (HIV), Epstein Barr Virus (EBV), Rous SarcomaVirus (RSV), and other retroviruses, and the thymidine kinase promoterof Herpes Simplex Virus. In one embodiment, the antibody orantigen-binding fragment thereof coding sequence is placed under controlof an inducible promoter such as the metallothionein promoter,tetracycline-inducible promoter, doxycycline-inducible promoter,promoters that contain one or more interferon-stimulated responseelements (ISRE) such as protein kinase R 2′,5′-oligoadenylatesynthetases, Mx genes, ADAR1, and the like.

Vectors described herein may contain one or more Internal Ribosome EntrySite(s) (IRES). Inclusion of an IRES sequence into fusion vectors may bebeneficial for enhancing expression of some proteins. In someembodiments the vector system will include one or more polyadenylationsites (e.g., SV40), which may be upstream or downstream of any of theaforementioned nucleic acid sequences. Vector components may becontiguously linked, or arranged in a manner that provides optimalspacing for expressing the gene products (i.e., by the introduction of“spacer” nucleotides between the ORFs), or positioned in another way.Regulatory elements, such as the IRES motif, may also be arranged toprovide optimal spacing for expression.

The vectors may comprise selection markers, which are well known in theart. Selection markers include positive and negative selection markers,for example, antibiotic resistance genes (e.g., neomycin resistancegene, a hygromycin resistance gene, a kanamycin resistance gene, atetracycline resistance gene, a penicillin resistance gene), glutamatesynthase genes, HSV-TK, HSV-TK derivatives for ganciclovir selection, orbacterial purine nucleoside phosphorylase gene for 6-methylpurineselection (Gadi et al., 7 Gene Ther. 1738-1743 (2000)). A nucleic acidsequence encoding a selection marker or the cloning site may be upstreamor downstream of a nucleic acid sequence encoding a polypeptide ofinterest or cloning site.

The vectors described herein may be used to transform various cells withthe genes encoding the described antibodies or antigen-bindingfragments. For example, the vectors may be used to generate antibody orantigen-binding fragment-producing cells. Thus, another aspect featureshost cells transformed with vectors comprising a nucleic acid sequenceencoding an antibody or antigen-binding fragment thereof thatspecifically binds GD2, such as the antibodies or antigen-bindingfragments described and exemplified herein.

Numerous techniques are known in the art for the introduction of foreigngenes into cells and may be used to construct the recombinant cells forpurposes of carrying out the described methods, in accordance with thevarious embodiments described and exemplified herein. The technique usedshould provide for the stable transfer of the heterologous gene sequenceto the host cell, such that the heterologous gene sequence is heritableand expressible by the cell progeny, and so that the necessarydevelopment and physiological functions of the recipient cells are notdisrupted. Techniques which may be used include but are not limited tochromosome transfer (e.g., cell fusion, chromosome mediated genetransfer, micro cell mediated gene transfer), physical methods (e.g.,transfection, spheroplast fusion, microinjection, electroporation,liposome carrier), viral vector transfer (e.g., recombinant DNA viruses,recombinant RNA viruses) and the like (described in Cline, 29 Pharmac.Ther. 69-92 (1985)). Calcium phosphate precipitation and polyethyleneglycol (PEG)-induced fusion of bacterial protoplasts with mammaliancells may also be used to transform cells.

Cells suitable for use in the expression of the antibodies orantigen-binding fragments described herein are preferably eukaryoticcells, more preferably cells of plant, rodent, or human origin, forexample but not limited to NSO, CHO, perC.6, Tk-tsl3, BHK, HEK293 cells,COS-7, T98G, CV-1/EBNA, L cells, C127, 3T3, HeLa, NS1, Sp2/0 myelomacells, and BHK cell lines, among others. In addition, expression ofantibodies may be accomplished using hybridoma cells. Methods forproducing hybridomas are well established in the art.

Cells transformed with expression vectors described herein may beselected or screened for recombinant expression of the antibodies orantigen-binding fragments described herein. Recombinant-positive cellsare expanded and screened for subclones exhibiting a desired phenotype,such as high level expression, enhanced growth properties, or theability to yield proteins with desired biochemical characteristics, forexample, due to protein modification or altered post-translationalmodifications. These phenotypes may be due to inherent properties of agiven subclone or to mutation. Mutations may be effected through the useof chemicals, UV-wavelength light, radiation, viruses, insertionalmutagens, inhibition of DNA mismatch repair, or a combination of suchmethods.

Once a cell expressing the desired protein is identified, it can beexpanded and selected. Transformed cells may be selected in a number ofways. For example, cells may be selected for expression of thepolypeptide of interest. Cells transformed with a vector that contains aselectable marker, such as production of fluorescent protein, may bepositively selected for expression of the marker. In other embodiments,the cells containing a vector with a drug resistance gene may bepositively selected for the ability to grow under selective conditions.

Kits

A kit is provided for inhibiting or reducing growth of cancer cells in apatient. Also provided are kits for identifying the presence ofdysplastic cells in vitro or in vivo.

The kits described herein may comprise an antibody, antigen-bindingfragments thereof, or an antibody composition described herein andinstructions for using the kit in a method for inhibiting or reducinggrowth of tumor cells in the patient or in a method for identifying thepresence of dysplastic cells, for example, in a biological sample. Thekit may comprise at least one chemotherapeutic or cytotoxic reagent. Thekit may comprise an antifolate compound. The kit may comprise at leastone diagnostic reagent. An example of a diagnostic reagent is adetectable label, for example but not limited to a radioactive,fluorescent, or chromophoric agent (e.g., ¹¹¹In-DOTA). The detectablelabel may comprise an enzyme. The kit may comprise instructions and ameans for administering the antibody or antibody composition, forexample, by injection.

Uses and Methods of Making BSA-Ganglioside Conjugates

Described herein are albumin-ganglioside conjugates and methods forproducing such conjugates. To determine whether an antibody, orantigen-binding fragment, of interest is effective in mediating bindingto a given antigen, one must characterize antibody binding. There arenumerous ways to characterize antibody binding, such as dot-blot,western blot, immunoprecipitation assay, ELISA, FACS analysis/Flowcytometry, and immunofluorescence detection of bound antibody. While avariety of assays are available to test antibody binding, the particularassay used must be selected with an understanding of the antibody'spurpose in mind. For example, if an antibody or antigen-binding fragmentthereof is to be used in a clinical setting, it should be characterizedby an assay that will present the antigen of interest in a form mostlikely to be found in vivo. In this instance, and in the case of acell-surface protein, flow cytometry may be a useful assay because itallows the interaction of the antibody or antigen-binding fragmentthereof to be assessed in the context of the native antigen.

One way to identify cells containing a vector that encodes anantigen-specific binding protein is using an Enzyme-Linked ImmunoSorbentAssay (ELISA) that makes use of the antigen of interest. There are twomain approaches used to perform an ELISA: an indirect ELISA and asandwich ELISA. To carry out an indirect ELISA, the antigen of interestis adsorbed or fixed to the surface of a microtiter plate and then theantibody or antigen-binding fragment thereof of interest is added to themicrotiter plate to detect the antigen. Conversely, a sandwich ELISAmakes use of an antibody known to be specific for the antigen ofinterest, in addition to the antibody being characterized. The knownantibody is used to coat the microtiter plate, such that it will bind,or capture, the antigen of interest when it is added to the microtiterplate. Once the antigen of interest has been captured, the antibody orantigen-binding fragment thereof of interest is added to the microtiterplate to assess its binding characteristics for the antigen. In eithercase, bound antibody or antigen-binding fragment thereof is typicallydetected by an enzyme-conjugated secondary antibody that is specific forthe antibody or antigen-binding fragment thereof of interest.

An ELISA useful for characterizing ganglioside-specific antibodies orantigen-binding fragments would be useful in determining the potentialapplications of such antibodies or antigen-binding fragments. The use ofgangliosides as ELISA antigens is complicated, however, by the fact thatfree gangliosides are poorly immunogenic (Jacques et al., 4 Org. Biomol.Chem. 142-154 (2006)).

Ganglioside Conjugates

Disclosed herein are ganglioside-albumin conjugates that not only retainthe antigenic properties of the conjugated ganglioside, but increase thestability of the ganglioside and allow better adherence to an ELISAmicrotiter plate. A ganglioside is conjugated to albumin throughreductive amination of the ganglioside by primary amines in albumin inthe presence of sodium cyanoborohydride. Conjugation proceeds throughSchiff base formation to the reducing end(s) of the carbohydrate moietyon the ganglioside, followed by reduction with sodium cyanoborohydride.Accordingly, some embodiments described herein include a gangliosideconjugated to a carrier protein via a reducing end(s) of thecarbohydrate moiety on the ganglioside. Some embodiments comprise aganglioside conjugated to a carrier protein, wherein the carrier is BSA.In some embodiments, the ganglioside is GD2. In some embodiments, theganglioside is GM3. In some embodiments, the ganglioside is GM2. Inanother embodiment, GD2 is conjugated to BSA. In some embodiments, thereductive amination of a ganglioside is catalyzed by sodiumcyanoborohydride.

In some embodiments the ganglioside-albumin conjugate retainsganglioside antigenicity such that a ganglioside-specific monoclonalantibody or antigen-binding fragment thereof can bind the conjugate. Insome embodiments the GD2-BSA conjugate retains GD2 antigenicity suchthat a GD2-specific monoclonal antibody or antigen-binding fragmentthereof can bind the conjugate. In some embodiments the GM2-BSAconjugate retains GM2 antigenicity such that a GM2-specific monoclonalantibody or antigen-binding fragment thereof can bind the conjugate. Insome embodiments the GM3-BSA conjugate retains GM3 antigenicity suchthat a GM3-specific monoclonal antibody or antigen-binding fragmentthereof can bind the conjugate.

The albumin-ganglioside conjugates described herein may be used in anELISA to characterize the specificity of an antibody or antigen-bindingfragment thereof for a particular ganglioside. In one aspect, thealbumin-ganglioside conjugates described provide an enhanced ability toadsorb the conjugated gangliosides to ELISA plates relative tounconjugated gangliosides. Once adsorbed, the conjugates may be used asganglioside antigens in a direct ELISA to characterize binding aspectsof ganglioside specific antibodies or antigen-binding fragments. In oneembodiment, the albumin-ganglioside conjugate may be a BSA-GD2conjugate. In one embodiment, the BSA-ganglioside conjugate may be aBSA-GM2 conjugate. In one embodiment, the BSA-ganglioside conjugate maybe a BSA-GM3 conjugate. Other such gangliosides may be conjugated to BSAusing the methods described and then used for the purposes describedherein. Such conjugates and uses thereof are within the scope of thisdisclosure.

Embodiments of the protein conjugates include the following.

Embodiment A1 provides a protein conjugate comprising albumin and aganglioside.

Embodiment A2 provides the conjugate of embodiment A1 wherein thealbumin is bovine serum albumin.

Embodiment A3 provides the conjugate of embodiment A1, wherein theganglioside is conjugated to albumin via a reducing end of thecarbohydrate moiety on the ganglioside.

Embodiment A4 provides the conjugate of embodiment A3 wherein thealbumin is bovine serum albumin.

Embodiment A5 provides the conjugate of embodiment A3 wherein theganglioside is GD2.

Embodiment A6 provides the conjugate of embodiment A1 wherein theconjugate is immunoreactive with a GD2-specific antibody.

Embodiment A7 provides the conjugate of embodiment A1, wherein theganglioside is GM2.

Embodiment A8 provides the conjugate of embodiment A7, wherein theconjugate is immunoreactive with a GM2-specific antibody.

Embodiment A9 provides the conjugate of embodiment A1, wherein theganglioside is GM3.

Embodiment A10 provides the conjugate of embodiment A9, wherein theconjugate is immunoreactive with a GM3-specific antibody.

Embodiment A11 provides a method of conjugating a ganglioside toalbumin, the method comprising reductively aminating a reducing end of acarbohydrate moiety on the ganglioside.

Embodiment A12 provides the method of embodiment A11, wherein thealbumin is bovine serum albumin.

Embodiment A13 provides the method of embodiment A11, wherein thereductive amination is catalyzed by sodium cyanoborohydride.

Embodiment A14 provides the method of embodiment A13, wherein theganglioside is GD2.

Embodiment A15 provides the method of embodiment A13, wherein theganglioside is GM2.

Embodiment A16 provides the method of embodiment A13, wherein theganglioside is GM3.

Cell Culture Compositions and Methods

Described herein are compositions and methods for culturing eukaryoticcells. The cells may be any type of eukaryotic cell, but, in oneembodiment, are preferably of mammalian origin. In some embodiments, thecells are Chinese Hamster ovary (CHO) cells. In some embodiments, theeukaryotic cells are genetically modified to produce IgM antibodies orantigen-binding fragments. The eukaryotic cells may be clinicalisolates, transformed with foreign DNA or RNA, immortalized,virus-infected, or modified by other generally known biological orchemical means. The eukaryotic cells may be grown in monolayers or insuspended culture, which may occur in a cell culture incubator,bioreactor, shake-flask, or other similar tissue culture device. Inaddition, the eukaryotic cells can be cultured at a temperature above32° C. and below 50° C., in between 2% to 8% CO₂, with or withoutshaking. In one embodiment, the cells can be cultured at a temperatureof 37° C., in 5% CO₂, with shaking at 120 rpm. The eukaryotic cells canbe cultured in tissue culture media, such as GIBCO-CD-CHO completemedium (1 L GIBCO-CD-CHO medium+25 μM MSX) or similar complete medium,in combination with the compositions and methods disclosed herein. Thecultured eukaryotic cells may be engineered to produce a foreignprotein, which the compositions and methods described herein can, butneed not necessarily, enhance.

Disclosed herein are compositions suitable for use with eukaryotic cellculture media. The composition includes, but is not limited to, variousessential and nonessential amino acids, for example, alanine, arginine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine, and valine; sugars,for example, glucose, fructose, mannose, and galactose; and vitamins,such as folic acid, vitamin B-12, vitamin D, and riboflavin that mayinfluence growth characteristics of cultured eukaryotic cells. Inembodiment B1, the composition includes glucose, glutamate, aspartate,serine, histidine, threonine, arginine, tyrosine, cysteine, valine,methionine, tryptophan, phenyalanine, isoleucine, leucine, lysine,proline, nicotinic acid amide, pyridoxine HCl, folic acid, vitamin B-12,riboflavin, and thiamine HCl.

In embodiment B2, the composition comprises the cell culture ofembodiment B1, wherein the concentration of glucose is 50 g/L, theconcentration of glutamate is from 2.5 to 3.75 g/L, the concentration ofaspartate is from 1.5 to 2.0 g/L, the concentration of serine is from0.3 to 0.5 g/L, the concentration of histidine is from 1.1 to 1.5 g/L,the concentration of threonine is from 2.0 to 3.0 g/L, the concentrationof arginine is from 1.0 to 1.5 g/L, the concentration of tyrosine isfrom 1.8 to 2.2 g/L, the concentration of cysteine is from 0.9 to 1.1g/L, the concentration of valine is from 1.0 to 3.0, the concentrationof methionine is from 0.8 to 1.2 g/L, the concentration of Tryptophan isfrom 0.5 to 0.8 g/L, the concentration of phenyalanine 1.3 to 1.7 g/L,the concentration of isoleucine is from 0.8 to 2.4 g/L, theconcentration of leucine is from 1.5 to 4.5 g/L, the concentration oflysine is from 3.5 to 5.0 g/L, the concentration of proline is from 0.5to 0.7 g/L, the concentration of nicotinic acid amide is from 30 to 40mg/L, the concentration of pyridoxine HCl is from 200 to 250 mg/L, theconcentration of folic acid 100 to 130 mg/L, the concentration ofvitamin B-12 20 to 40 mg/L, the concentration of riboflavin is from 20to 40 mg/L, and the concentration of thiamine HCl is from 100 to 150mg/L.

Also disclosed herein are methods for culturing eukaryotic cells inwhich a composition described in the preceding paragraph may be used toenhance the viability of eukaryotic cells. For example, the compositionsmay be added to cultured CHO cells and to cultured IgM-producingeukaryotic (e.g., CHO) cells. The composition may be used to enhance theviability of eukaryotic tissue culture cells by adding the compositionto growth media, as a media supplement, either before or after growthmedia is applied to eukaryotic cells. Alternatively, growth mediacomponents may be added to the composition to produce a growth mediathat contains the composition.

Embodiment B3 provides a method of culturing eukaryotic cells comprisingadding to the cells a primary tissue culture media and thensupplementing the primary tissue culture media with a cell culturecomposition comprising, glucose, glutamate, aspartate, serine,histidine, threonine, arginine, tyrosine, cysteine, valine, methionine,tryptophan, phenyalanine, isoleucine, leucine, lysine, proline,nicotinic acid amide, pyridoxine HCl, folic acid, vitamin B-12,riboflavin, and thiamine HCl.

Embodiment B4 provides the method of embodiment B3 wherein the volume ofthe cell culture composition that is added to the primary tissue culturemedia is between 1.0% and 20% of the volume of the primary tissueculture media.

Embodiment B5 provides the method of embodiment B3, wherein the cellculture composition is not added to the primary tissue culture mediabefore the third day following addition of the primary tissue culturemedia.

Embodiment B6 provides the method of embodiment B3, wherein theeukaryotic cells have been transformed to produce one or more proteins.

Embodiment B7 provides the method of embodiment B6, wherein the one ormore proteins is an antibody, or an antigen-binding fragment thereof.

Embodiment B8 provides the method of embodiment B7 wherein the antibodyis an IgM.

Embodiment B9 provides the method of embodiment B3, wherein the cellculture composition is added to the primary tissue culture media onconsecutive days.

In another embodiment, the composition may be used to increase theamount of protein produced by a eukaryotic cell that is geneticallymodified to produce a recombinant protein. For example, the compositionmay be added to cultured cells engineered to express a recombinant humanantibody or antigen-binding fragment thereof (e.g., cells modified toexpress IgM and more specifically CHO cells modified to express IgM).The composition may be used to increase recombinant protein expressionby adding the composition to growth media, as a media supplement, eitherbefore or after growth media is applied to eukaryotic cells.Alternatively, growth media components may be added to the compositionto produce a growth media that contains the composition.

The composition may be used as growth media or as a supplement forgrowth media. As a supplement the composition may be added to cellculture media either before or after the culture media is added totissue culture containers. The composition described herein may be usedto supplement eukaryotic cell culture media to a total concentration of1.5% to 2.5%. These same concentrations may be used to produce cellculture media already containing the composition.

Also featured are methods for increasing the amount of protein producedby a eukaryotic cell that is genetically modified to produce arecombinant protein by adding valeric acid to cell culture media. Forexample, the valeric acid may be added to media for cultured CHO,hybridoma, and NS0 cells engineered to express a recombinant humanantibody or antigen-binding fragment. In one embodiment, valeric acidmay be added to cell culture media to a final concentration from 0.1 mMto 10 mM. In one embodiment, valeric acid may be added at specificpoints in the cell growth period, for example, valeric acid may be addedto the culture media on specific days following revival of cell stocksor after cell cultures are split.

Embodiment B10 provides a method of enhancing protein production bycells, comprising supplementing the growth media of the cells withvaleric acid.

Embodiment B11 provides the method of embodiment B10, wherein the cellsare antibody-producing cells.

Embodiment B12 provides the method of embodiment B10, wherein the growthmedia is supplemented to have a concentration of valeric acid from 0.1mM to 10 mM.

Antibody Isolation or Purification

The antibodies or antigen-binding fragments described herein can beseparated from a substantial portion of the cellular growth media fromwhich they are recovered to allow for a more purified or isolated formof the antibody or antigen-binding fragment. Described herein aremethods to purify or isolate antibodies or antigen-binding fragments,such as human IgM antibodies or antigen-binding fragments, from asolution, e.g., conditioned culture supernatant (CCS). Increasing thepurity of a solution containing the antibodies or antigen-bindingfragments may be accomplished in a number of ways, including, but notlimited to, dialysis, size exclusion chromatography, centrifugation,ion-exchange chromatography, gradient centrifugation, filtration with asize-exclusion filter, affinity chromatography, immunoaffinitychromatography, and high performance liquid chromatography. Thedescribed antibodies or antigen-binding fragments may also be modified,genetically or chemically, to include an affinity tag, such aspolyhistidine, that may be used to increase antibody or antigen-bindingfragment thereof purity through affinity purification techniques.

Accordingly, some embodiments include an antibody or antigen-bindingfragment thereof that is separated from a solution by affinitychromatography. Some embodiments comprise an antibody or antigen-bindingfragment thereof that is substantially separated from a solution byaffinity chromatography, wherein affinity for the antibody orantigen-binding fragment thereof is mediated by protein A. Someembodiments comprise an antibody or antigen-binding fragment thereofthat is substantially separated from a solution by ion-exchangechromatography. Some embodiments comprise an antibody or antigen-bindingfragment thereof that is substantially separated from a solution bycation-exchange chromatography, wherein the cation-exchangechromatography column comprises an acrylamide-dextran copolymer resin.Some embodiments comprise an antibody or antigen-binding fragmentthereof that is substantially separated from a solution byhydroxyapatite chromatography. Some embodiments comprise an antibody orantigen-binding fragment thereof that is substantially separated from asolution by hydroxyapatite chromatography, wherein the hydroxyapatitechromatography column incorporates a ceramic calcium phosphate resin.

The process by which the described antibodies or antigen-bindingfragments can be purified or isolated may vary as the requirements forthe resulting antibody or antigen-binding fragment thereof may differdepending on the application for which the antibodies or antigen-bindingfragments will be used. For example, antibodies or antigen-bindingfragments for use in a pharmaceutical composition would requirestringent purification, while antibodies or antigen-binding fragmentsfor use in an in vitro diagnostic assay can be purified to a lesserdegree. Accordingly some embodiments include contacting an antibody orantigen-binding fragment-containing sample with a protein A matrix. Someembodiments include contacting an antibody or antigen-bindingfragment-containing sample with an ion-exchange matrix. In oneembodiment, the ion-exchange matrix can be a cation-exchangechromatography matrix. In a more preferable embodiment thecation-exchange chromatography matrix can include acrylamide-dextrancopolymer resin. Some embodiments include contacting an antibody orantigen-binding fragment-containing sample with an hydroxyapatitematrix. In one embodiment, the hydroxyapatite matrix can include acalcium phosphate resin.

Serial affinity chromatography is a process that yields an isolated orpurified protein. The process may employ two or more rounds of differingaffinity chromatography techniques to produce a purified or isolatedprotein of interest (Friedrichs and Grose, 49 J. Virol. 992 (1984)).Accordingly, in some embodiments an antibody or antigen-bindingfragment-containing sample may be contacted with a protein A matrixunder conditions that promote binding of antibodies or antigen-bindingfragments to the matrix; the matrix may be washed to remove unboundproteins; the material bound by the protein A matrix may be eluted; theeluted material may be contacted by a cation-exchange chromatographymatrix under conditions that promote binding of antibodies orantigen-binding fragments to the matrix; the matrix may be washed toremove unbound proteins; the material bound by the cation-exchangechromatography matrix may be eluted; the eluted material may becontacted by a hydroxyapatite resin under conditions that promotebinding of antibodies or antigen-binding fragments to the matrix; thematrix may be washed to remove unbound proteins; and the bound materialmay be eluted. In addition, optional steps may be added to variouspoints in this process to allow for more stringent purification of theantibody or antigen-binding fragment thereof. For example, the antibodyor antigen-binding fragment-containing solution may be supplemented withdetergent, such as Triton®-X 100 or Tween®80, to inactivate microbes,such as bacteria, viruses, and parasites.

Affinity-based chromatography of proteins is a multi-step process thatgenerally involves equilibrating a chromatography column, contacting asample solution with the matrix of a column, washing away the unboundmaterial in the column, and eluting the desired material. This generalprocess may be repeated one or more times, under varying conditions, toincrease the purity of a sample, as needed. In some embodiments aprotein A matrix may be used to contact the sample to be purified. Inone embodiment, the protein A matrix may be porous, having pores rangingfrom 1000 to 5000 angstroms in diameter. In another embodiment theprotein A matrix has pores with an average diameter of 3000 angstroms.

In some embodiments the affinity matrix is washed and equilibratedbefore use. For example, the affinity matrix may be washed with purifiedwater to remove any contaminants. In some embodiments the affinitymatrix may be washed with 3 to 10 column volumes of purified water. Inanother embodiment the affinity matrix may be washed with 5 columnvolumes of purified water. In some embodiments the affinity matrix maybe washed with at least one acidic buffer. For example, the affinitymatrix may be washed with 1 to 5 column volumes of 20 mM HCl at pH 1.5.In another embodiment the affinity matrix is washed with 3 columnvolumes of 20 mM HCl at pH 1.5.

In addition, the affinity matrix may be washed with at least oneadditional buffer, such as 6 M guanidine-HCl. Before contacting theaffinity matrix with a sample the matrix may be equilibrated with amildly basic or neutral pH buffer. In one embodiment, the affinitymatrix may be equilibrated with 2 to 8 column volumes of a buffer at pH7.5. In another embodiment the affinity matrix may be equilibrated with5 column volumes of a 10 mM sodium phosphate buffer containing 200 mMNaCl and 0.01% Tween®-80, at pH 7.5. Sodium phosphate buffers describedherein may be prepared from a mixture of monobasic and dibasic sodiumphosphate. Mixed in the correct ratios, these solutions may yieldphosphate buffers within the range of pH 4 to pH 10.

Samples to be purified or isolated via contacting the affinity matrixshould be substantially free of large particulate matter. Suchparticulate matter may be removed in a variety of ways, such as bycentrifugation or filtration. In another embodiment the conditionedculture supernatant (CCS) described herein is made substantially free oflarge particulate matter before contact with an affinity matrix. In yetanother embodiment the CCS is filtered to remove particulate matter. Afilter with an appropriate pore size may be used, such as a filterhaving pores with an average diameter of 1 μm or a filter having poreswith an average diameter of 0.75 μm or a filter having pores with anaverage diameter of 0.5 μm or a filter having pores with an averagediameter of 0.22 μm, or a filter having pores with an average diameterof 0.1 μm.

Before contacting the affinity matrix with a solution containing asample to be purified or isolated, the solution may be treated withdetergent to inactivate at least one microbial contaminant, such asbacteria, viruses, and parasites. In one embodiment, the CCS issupplemented with detergent to inactivate at least one microbialcontaminant. In one embodiment, the detergent is Triton® X-100. Theconcentration of detergent added to the solution containing a sample tobe purified or isolated may vary to meet the needs of a particularpurification scheme. In one embodiment, the solution containing a sampleto be purified or isolated may have 10% detergent. In anotherembodiment, the solution containing a sample to be purified or isolatedmay have 7% detergent. In one embodiment, the solution containing asample to be purified or isolated may have 5% detergent. In oneembodiment, the solution containing a sample to be purified or isolatedmay have 3% detergent. In one embodiment, the solution containing asample to be purified or isolated may have 2% detergent. In oneembodiment, the solution containing a sample to be purified or isolatedmay have 1% detergent. In one embodiment, the solution containing asample to be purified or isolated may have 0.5% detergent. In oneembodiment, the solution containing a sample to be purified or isolatedmay have 0.1% detergent.

Once applied to the affinity matrix, the sample may be processed at adesired flow rate, for example, to facilitate processing or binding ofthe sample. While any flow rate may be used, common flow rates arebetween 1 and 200 cm/h. In some embodiments the flow rate of the samplemay be 1 cm/h. In other embodiments the flow rate of the sample may be10 cm/h. In one embodiment, the flow rate of the sample may be 25 cm/h.In one embodiment, the flow rate of the sample may be 50 cm/h. Inanother embodiment the flow rate of the sample may be 76 cm/h. Inanother embodiment the flow rate of the sample may be 100 cm/h. In someembodiments the flow rate of the sample may be 125 cm/h. In anotherembodiment the flow rate of the sample may be 150 cm/h. In oneembodiment, the flow rate of the sample may be 175 cm/h. In oneembodiment, the flow rate of the sample may be 200 cm/h.

After the affinity matrix has been contacted by the sample material,unbound sample material may be removed by washing with a buffer. In oneembodiment, the affinity matrix may be washed with a buffer with anacidic pH. In one embodiment, the affinity matrix may be washed with abuffer with a neutral pH. In one embodiment, the affinity matrix may bewashed with a buffer with a basic pH. In another embodiment the affinitymatrix may be washed with 5 to 15 column volumes of a 10 mM sodiumphosphate buffer containing 200 mM NaCl and 0.01% Tween®-80, at pH 7.5.

After unbound proteins have been removed from the affinity matrix bywashing, proteins bound to the affinity matrix may be eluted by washingthe affinity matrix with an elution buffer. The elution buffer should beselected carefully to assure that it will disrupt the interactionbetween the protein of interest, e.g., a human IgM antibody, and thematrix, but will not denature or otherwise deteriorate the condition ofthe protein of interest. In one embodiment, the affinity matrix may bewashed with a buffer with an acidic pH to elute the protein of interest.In one embodiment, the affinity matrix may be washed with a buffer witha neutral pH to elute the protein of interest. In one embodiment, theaffinity matrix may be washed with a buffer with a basic pH to elute theprotein of interest. Some embodiments include a buffer having sodiumphosphate and magnesium chloride for eluting material bound to achromatography column, where up to 10 column volumes of this buffer areused to elute bound material from the column. In some embodiments, abuffer having between 5 and 10 mM sodium phosphate and 1 and 5 Mmagnesium chloride may be used for eluting material bound to an affinitymatrix. Another embodiment, includes 3 column volumes of a buffercomprising 5 mM sodium phosphate and 3 M magnesium chloride to elutematerial bound to an affinity matrix. In some embodiments IgM may beeluted from a protein A matrix using a 5 mM sodium phosphate buffersupplemented with 3 M MgCl₂ at pH 6.8.

Disclosed herein are methods for purifying or isolating proteins, e.g.,IgM antibodies or antigen-binding fragments, using ion-exchangechromatography. Ion-exchange chromatography of proteins is a multi-stepprocess that generally involves equilibrating a chromatography column,contacting a sample solution with the matrix of a column, washing awaythe unbound material in the column, and eluting the desired material.This general process may be repeated one or more times, under varyingconditions, to increase the purity of a sample, as needed. In someembodiments the cation exchange chromatography matrix may be anacrylamide-dextran copolymer resin. In one embodiment, the cationexchange chromatography matrix may be MacroCap™ SP resin.

In some embodiments the ion-exchange matrix is washed and equilibratedbefore use. For example, the ion-exchange matrix may be washed withpurified water to remove any contaminants. In one embodiment, theion-exchange matrix may be washed with 1 to 10 column volumes ofpurified water. In one embodiment, the ion-exchange matrix may be washedwith 2 column volumes of purified water. In some embodiments theion-exchange matrix may be washed with at least one buffer. For example,the ion-exchange matrix may be washed with 1 to 5 column volumes of abuffer having 0.5 M NaOH. In another embodiment the ion-exchange matrixis washed with 3 column volumes of a buffer having 0.5 M NaOH. In someembodiments the ion-exchange matrix is washed with 3 column volumes of abuffer having 2 M NaCl. Before contacting the ion-exchange matrix with asample the matrix may be equilibrated with an acidic, basic, or neutralpH buffer. In one embodiment, the ion-exchange matrix may beequilibrated with 2 to 8 column volumes of a buffer at pH 6.8. In oneembodiment, the ion-exchange matrix may be equilibrated with 5 columnvolumes of a 10 mM sodium phosphate buffer, containing 75 mM NaCl and0.01% Tween®-80, at pH 6.8.

Samples to be purified or isolated via contacting the ion-exchangematrix should be substantially free of large particulate matter. Suchparticulate matter may be removed in a variety of ways, such as bycentrifugation or filtration. In some embodiments the conditionedculture supernatant (CCS) described herein is made substantially free oflarge particulate matter before having contact with an ion-exchangematrix. In another embodiment the CCS is filtered to remove particulatematter. A filter with any pore size may be used, such as a filter havingpores with an average diameter of 1 μm or a filter having pores with anaverage diameter of 0.75 μm or a filter having pores with an averagediameter of 0.5 μm or a filter having pores with an average diameter of0.22 μm, or a filter having pores with an average diameter of 0.1 μm.

Before contacting the ion-exchange matrix with a solution containing asample to be purified, the solution may be treated with detergent toinactivate at least one microbial contaminant, such as bacteria,viruses, and parasites, or to help maintain the target protein in asoluble state. In some embodiments the sample may be supplemented withdetergent to inactivate at least one microbial contaminant or to helpmaintain the target protein in a soluble state. In one embodiment, thedetergent is Triton® X-100. In one embodiment, the detergent isTween®-80. The concentration of detergent added to the solutioncontaining a sample to be purified or isolated may vary to meet theneeds of a particular purification scheme. In one embodiment, thesolution containing a sample to be purified or isolated may have 10%detergent. In one embodiment, the solution containing a sample to bepurified or isolated may have 7% detergent. In one embodiment, thesolution containing a sample to be purified or isolated may have 5%detergent. In one embodiment, the solution containing a sample to bepurified or isolated may have 3% detergent. In one embodiment, thesolution containing a sample to be purified or isolated may have 2%detergent. In one embodiment, the solution containing a sample to bepurified or isolated may have 1% detergent. In one embodiment, thesolution containing a sample to be purified or isolated may have 0.5%detergent. In one embodiment, the solution containing a sample to bepurified or isolated may have 0.1% detergent.

Once applied to the ion-exchange matrix, the sample may be processed ata desired flow rate, for example, to facilitate processing or binding ofthe sample. While any flow rate may be used, common flow rates arebetween 1 and 200 cm/h. In one embodiment, the flow rate of the samplemay be 1 cm/h. In one embodiment, the flow rate of the sample may be 10cm/h. In one embodiment, the flow rate of the sample may be 25 cm/h. Inone embodiment, the flow rate of the sample may be 50 cm/h. In oneembodiment, the flow rate of the sample may be 76 cm/h. In oneembodiment, the flow rate of the sample may be 100 cm/h. In oneembodiment, the flow rate of the sample may be 125 cm/h. In oneembodiment, the flow rate of the sample may be 150 cm/h. In oneembodiment, the flow rate of the sample may be 175 cm/h. In anotherembodiment the flow rate of the sample may be 200 cm/h.

After the ion-exchange matrix has been contacted by the sample material,unbound sample material may be removed by washing with a buffer. In oneembodiment, the ion-exchange matrix may be washed with a buffer with anacidic pH. In one embodiment, the ion-exchange matrix may be washed witha buffer with a neutral pH. In one embodiment, the ion-exchange matrixmay be washed with a buffer with a basic pH. In one embodiment, theion-exchange matrix may be washed with 5 to 15 column volumes of abuffer containing 10 mM sodium phosphate, 75 mM NaCl, and 0.01%Tween®-80, at pH 6.8.

After unbound proteins have been removed from the ion-exchange matrix bywashing, proteins bound to the ion-exchange matrix may be eluted bywashing the ion-exchange matrix with an elution buffer. The elutionbuffer should be selected carefully to assure that it will disrupt theinteraction between the protein of interest, e.g., a human IgM antibody,and the matrix, but will not denature or otherwise deteriorate thecondition of the protein of interest. In one embodiment, theion-exchange matrix may be washed with a buffer with an acidic pH toelute the protein of interest. In one embodiment, the ion-exchangematrix may be washed with a buffer with a neutral pH to elute theprotein of interest. In one embodiment, the ion-exchange matrix may bewashed with a buffer with a basic pH to elute the protein of interest.Some embodiments include a buffer having sodium phosphate and sodiumchloride for eluting material bound to a chromatography column, where upto 10 column volumes of this buffer are used to elute bound materialfrom the column. In some embodiments, a buffer having between 5 and 50mM sodium phosphate and 150 and 500M sodium chloride may be used foreluting material bound to an ion-exchange matrix. Another embodimentincludes 4 column volumes of a buffer comprising 10 mM sodium phosphatebuffer containing 200 mM NaCl to elute material bound to an ion-exchangematrix. In some embodiments IgM may be eluted from a MacroCap™ SP matrixusing a 10 mM sodium phosphate buffer containing 200 mM NaCl and 0.01%Tween®-80, at pH 6.8.

Disclosed herein are methods for purifying or isolating proteins, e.g.,IgM antibodies or antigen-binding fragments, using hydroxyapatitechromatography. Hydroxyapatite chromatography of proteins is amulti-step process that generally involves equilibrating achromatography column, contacting a sample solution with the matrix of acolumn, washing away the unbound material in the column, and eluting thedesired material. This general process may be repeated one or moretimes, under varying conditions, to increase the purity of a sample, asneeded. In some embodiments the hydroxyapatite chromatography matrix maybe a calcium phosphate matrix. In one embodiment, the hydroxyapatitechromatography matrix may be CHT® II Ceramic Hydroxyapatite, 80 μm beadsize.

In some embodiments the hydroxyapatite matrix is hydrated before use. Inone embodiment, the hydroxyapatite matrix is hydrated with a solutionhaving 200 mM potassium phosphate, at pH 9.0, using 0.54 g of dry matrixfor each mL of desired column-bed volume. Before contacting thehydroxyapatite matrix with a sample the matrix may be equilibrated withan acidic, basic, or neutral pH buffer. In one embodiment, thehydroxyapatite matrix may be equilibrated with 2 to 8 column volumes ofa buffer at pH 6.8. In another embodiment the hydroxyapatite matrix maybe equilibrated with 5 column volumes of a buffer containing 10 mMsodium phosphate, 100 mM NaCl, and 0.01% Tween®-80, at pH 6.8.

Samples to be purified or isolated via contacting the hydroxyapatitematrix should be substantially free of large particulate matter. Suchparticulate matter may be removed in a variety of ways, such as bycentrifugation or filtration. In one embodiment, the conditioned culturesupernatant (CCS) is made substantially free of large particulate matterbefore having contact with a hydroxyapatite matrix. In one embodiment,the CCS is filtered to remove particulate matter. A filter with any poresize may be used, such as a filter having pores with an average diameterof 1 μm or a filter having pores with an average diameter of 0.75 μm ora filter having pores with an average diameter of 0.5 μm or a filterhaving pores with an average diameter of 0.22 μm, or a filter havingpores with an average diameter of 0.1 μm.

Before contacting the hydroxyapatite matrix with a solution containing asample to be purified, the solution may be treated with detergent toinactivate at least one microbial contaminant, such as bacteria,viruses, and parasites, or to help maintain the target protein in asoluble state. In some embodiments the sample may be supplemented withdetergent to inactivate at least one microbial contaminant or to helpmaintain the target protein in a soluble state. In one embodiment, thedetergent is Triton® X-100. In one embodiment, the detergent isTween®-80. The concentration of detergent added to the solutioncontaining a sample to be purified or isolated may vary to meet theneeds of a particular purification scheme. In one embodiment, thesolution containing a sample to be purified or isolated may have 10%detergent. In one embodiment, the solution containing a sample to bepurified or isolated may have 7% detergent. In one embodiment, thesolution containing a sample to be purified or isolated may have 5%detergent. In one embodiment, the solution containing a sample to bepurified or isolated may have 3% detergent. In one embodiment, thesolution containing a sample to be purified or isolated may have 2%detergent. In one embodiment, the solution containing a sample to bepurified or isolated may have 1% detergent. In one embodiment, thesolution containing a sample to be purified or isolated may have 0.5%detergent. In one embodiment, the solution containing a sample to bepurified or isolated may have 0.1% detergent.

Once applied to the hydroxyapatite matrix, the sample may be processedat a desired flow rate, for example, to facilitate processing or bindingof the sample. While any flow rate may be used, common flow rates arebetween 1 and 200 cm/h. In one embodiment, the flow rate of the samplemay be 1 cm/h. In one embodiment, the flow rate of the sample may be 10cm/h. In one embodiment, the flow rate of the sample may be 25 cm/h. Inone embodiment, the flow rate of the sample may be 50 cm/h. In oneembodiment, the flow rate of the sample may be 76 cm/h. In oneembodiment, the flow rate of the sample may be 100 cm/h. In oneembodiment, the flow rate of the sample may be 125 cm/h. In oneembodiment, the flow rate of the sample may be 150 cm/h. In oneembodiment, the flow rate of the sample may be 175 cm/h. In oneembodiment, the flow rate of the sample may be 200 cm/h.

After the hydroxyapatite matrix has been contacted by the samplematerial, unbound sample material may be removed by washing with abuffer. In one embodiment, the hydroxyapatite matrix may be washed witha buffer with an acidic pH. In one embodiment, the hydroxyapatite matrixmay be washed with a buffer with a neutral pH. In one embodiment, thehydroxyapatite matrix may be washed with a buffer with a basic pH. Inone embodiment, the hydroxyapatite matrix may be washed with 5 to 15column volumes of a buffer containing 10 mM sodium phosphate, 100 mMNaCl, and 0.01% Tween®-80, at pH 6.8.

After unbound proteins have been removed from the hydroxyapatite matrixby washing, proteins bound to the hydroxyapatite matrix may be eluted bywashing the hydroxyapatite matrix with an elution buffer. The elutionbuffer should be selected carefully to assure that it will disrupt theinteraction between the protein of interest, e.g., a human IgM antibody,and the matrix, but will not denature or otherwise deteriorate thecondition of the protein of interest. In one embodiment, thehydroxyapatite matrix may be washed with a buffer with an acidic pH toelute the protein of interest. In one embodiment, the hydroxyapatitematrix may be washed with a buffer with a neutral pH to elute theprotein of interest. In one embodiment, the hydroxyapatite matrix may bewashed with a buffer with a basic pH to elute the protein of interest.Some embodiments include a buffer having sodium phosphate and sodiumchloride for eluting material bound to a hydroxyapatite column, where upto 10 column volumes of this buffer are used to elute bound materialfrom the column. In some embodiments, a buffer having between 150 and500 mM sodium phosphate and 50 and 200 M sodium chloride may be used foreluting material bound to a hydroxyapatite matrix. In one embodiment, 4column volumes of a buffer comprising 175 mM sodium phosphate buffercontaining 100 mM NaCl may be used to elute material bound to anhydroxyapatite matrix. In another embodiment IgM may be eluted from aCHT® II Ceramic Hydroxyapatite matrix using a buffer containing 175 mMsodium phosphate, 100 mM NaCl, and 0.01% Tween®-80, at pH 6.8.

Buffers of varying composition, with respect to salts used, saltconcentration, detergents used, detergent concentration, pH, and thelike, may be used to substantially perform the methods described herein.Accordingly, such variations are contemplated to be within the scope ofthe disclosure provided, which is not intended to be exclusive orlimiting.

Embodiment C1 provides a method of purifying or isolating a protein, themethod comprising applying a solution comprising the protein to anaffinity chromatography column, applying eluate from the affinitychromatography column to a cation exchange chromatography column, andapplying eluate from the cation exchange chromatography column to ahydroxyapatite chromatography column, and obtaining the eluate from thehydroxyapatite chromatography column.

Embodiment C2 provides the method of embodiment C1, wherein the affinitychromatography column comprises at least one substrate coated withprotein A.

Embodiment C3 provides the method of embodiment C2, wherein the at leastone substrate coated with protein A is porous.

Embodiment C4 provides the method of embodiment C3, wherein the poroussubstrate comprises pores of 3000 angstroms.

Embodiment C5 provides the method of embodiment C1, wherein the at leastone substance coated with protein A binds a protein.

Embodiment C6 provides the method of embodiment C5, wherein the proteinbound by at least one substance coated with protein A is eluted with asolution comprising magnesium chloride.

Embodiment C7 provides the method of embodiment C6, wherein the solutioncomprising magnesium chloride is a solution of 1M to 5M magnesiumchloride.

Embodiment C8 provides the method of embodiment C6, wherein the solutioncomprising magnesium chloride is a solution of 3M magnesium chloride.

Embodiment C9 provides the method of embodiment C5, wherein the proteinbound by the at least one substance coated with protein A is anantibody.

Embodiment C10 provides the method of embodiment C9, wherein theantibody is IgM.

Embodiment C11 provides the method of embodiment C11, wherein the cationexchange chromatography column comprises an acrylamide-dextran copolymerresin.

Embodiment C12 provides the method of embodiment C11, wherein theacrylamide-dextran copolymer resin binds a protein.

Embodiment C13 provides the method of embodiment C12, wherein theprotein bound by the acrylamide-dextran copolymer resin is eluted with asolution comprising 10 mM sodium phosphate, 200 mM sodium chloride, and0.01% polysorbate 80.

Embodiment C14 provides the method of embodiment C13, wherein theprotein bound by the acrylamide-dextran copolymer resin is an antibody.

Embodiment C15 provides the method of embodiment C14, wherein theantibody is IgM.

Embodiment C16 provides the method of embodiment C1, wherein thehydroxyapatite chromatography column comprises a calcium phosphateresin.

Embodiment C17 provides the method of embodiment C16, wherein thecalcium phosphate resin binds a protein.

Embodiment C18 provides the method of embodiment C17, wherein theprotein bound by the calcium phosphate resin is eluted with a solutioncomprising 175 mM sodium phosphate, 100 mM sodium chloride, and 0.01%polysorbate 80.

Embodiment C19 provides the method of embodiment C18, wherein theprotein bound by the calcium phosphate resin is an antibody.

Embodiment C20 provides the method of embodiment C19, wherein theantibody is IgM.

Embodiment C21 provides a method of purifying or isolating a protein,comprising

-   -   a. applying a solution containing the protein to an affinity        chromatography column comprising at least one substrate coated        with protein A, and eluting any proteins bound by the at least        one substance coated with protein A with a solution comprising        3M magnesium chloride;    -   b. applying the eluate from the affinity chromatography column        to a cation exchange chromatography column comprising an        acrylamide-dextran copolymer resin, and eluting any proteins        bound by the acrylamide-dextran copolymer resin with a solution        comprising 10 mM sodium phosphate, 200 mM sodium chloride, and        0.01% polysorbate 80;    -   c. applying the eluate from the cation exchange chromatography        column to a hydroxyapatite comprising a calcium phosphate resin,        and eluting any proteins bound by the calcium phosphate resin        with a solution comprising 175 mM sodium phosphate, 100 mM        sodium chloride, and 0.01% polysorbate 80.

The following examples are provided to describe the embodimentsdescribed herein with greater detail. They are intended to illustrate,not to limit, the embodiments.

EXAMPLE 1 GD-2 Specificity of CV-3-Derived IgM

A pool of human lymphoblast cells transformed with Epstein-Barr Virus(HLP) (Cahan, et al.) was tested for the ability to produce GD2-specificIgM antibodies. The cells were cultured in RMPI complete media (10%heat-inactivated FBS, 1% L-Glu, 1% antibiotics (Sigma)). Spent growthmedia was collected at passages two through four and assayed for IgMconcentration and reactivity with GD2. The HLP produced 6-8 μg/mL IgM,(quantified by ELISA), after a culture of 2-5×10⁵ cells/mL was grown for3 days.

To determine whether the IgM produced by the HLP was specific for GD2,spent media from each culture was analyzed by ELISA. Briefly,gangliosides were coated to ELISA plates by evaporating 200 μl ofethanol containing 25 ng of GD2, GD1a, GM2 or GM3. Plates were thenblocked and binding of IgM-containing cell culture media or human IgMwere assessed according to regular ELISA procedures. Spent media fromthe HLP (concentrated either 8.1-fold or 14.2-fold) containedGD2-reactive IgM (FIG. 1A); however, the antibodies also recognizedgangliosides GD1a, GM2, and GM3 (FIG. 1B). These data indicate that IgMproduced by the HLP is polyreactive.

FACS analysis was conducted to determine whether IgM produced by the HLPcould bind GD2 expressed on the surface of melanoma cells. Briefly,1205LU cells, a human melanoma cell line that expresses GD2 on itssurface, were incubated with spent media from HLP cultures, washed, andthen labeled with secondary antibody FITC-goat-anti-human Ig (JacksonLaboratories). As shown in FIG. 2, only culture media from very earlypassages of the HLP (passage 2) produced IgM that could recognize cellsurface GD2 (FIG. 2B). However, this activity was quickly lost aftersubculturing the HLP for a few weeks (FIG. 2C). These results suggestthat the cells producing specific GD2-reactive IgM are slow growers inthe HLP and are quickly overpopulated by non-producers or nonspecificantibody producers, or that the production of this IgM is not stable.The murine anti-GD2 IgM antibody MAb-126 (ATCC# HB-8568™) was used as apositive control and demonstrated robust GD2-specific binding (FIG. 2D).

EXAMPLE 2 Hybridomas Derived from the HLP Produce GD-2 Specific IgM thatMediates CDC Activity on GD2-Positive Cells

In order to generate EBV-null hybridoma lines that produce anti-GD2specific IgM, cells from early passages of the HLP were fused withfusion partners A6 (ATCC CRL-8192) or K6H6/B5 (ATCC CRL-1823) to formhybridomas. Due to the apparently low abundance of GD2-specificsubclones in the HLP, 80,000 clones were screened by GD2-specific ELISA.Approximately 5-10% of antibody preparations from these hybridoma linesshowed positive GD2 reactivity. However, over 90% of the GD2 positiveclones showed reactivity to multiple gangliosides when screened forspecificity using a panel of gangliosides (GD1, GM2 and GM3) (FIG.3A-clones 5D7 and 10B4). Moreover, the majority of polyreactiveantibodies cross-reacted to a panel of non-related control proteins(data not shown). These results suggest that the lymphoblast cellsproducing GD2-specific IgM are rare in the HLP.

Subcloning and screening efforts led to the isolation of GD2-specificIgM-producing hybridoma cell lines. Two GD2 specific hybridoma lines,3B2 and 1470, were isolated. Cultures of both cell lines were seeded at0.3-0.4e⁶/mL of cells of seeding density and split every 3 to 4 days,which yielded 3-8 μg/mL of IgM in spent medium. Ganglioside-specificELISA revealed positive reactivity with GD2 only and no reactivity tothree control gangliosides (GD1a, GM2, and GM3) (FIGS. 3A and B),suggesting that these cells produce GD2-specific antibodies. Inaddition, FACS analysis showed positive staining of 1205LU humanmelanoma cells with 3B2 and 1470 antibodies, suggesting that theseantibodies can recognize GD2 on the cell surface (FIG. 4). Molecularanalyses of clones 3B2 and 1470 were performed to determine cloneorigin. Light chain specific ELISA showed that both clones secreted IgMcontaining a kappa light chain (data not shown). Sequence analysis ofcDNA from these cells revealed identical heavy and light chainsequences, suggesting that the two clones were derived from the same HLPlymphoblast clone (data not shown).

Partially purified antibody from the 3B2 culture was used to assess theability of this antibody to mediate complement-dependent cytotoxicity(CDC) on GD2 expressing melanoma cell lines, as this activity is largelydependent on the conformation that the antibody assumes when bound toits antigen (Janeway et al., Immunobiology, 9-12 (5^(th) ed. 2001)).Melanoma target cell lines (GD2-positive cell lines LF0023 and M14, andGD2-negative cell lines PM0496 and JS0592) were cultured in RPMI1640supplemented with 10% FBS, 2 mM L-Glutamine, non-essential amino acids,and 6 μM HEPES and harvested with trypsin before use. Target cells wereincubated with 3B2 IgM-containing supernatant in the presence of 20%human serum. After incubation for 1 hour at 37° C., live cells wereidentified with Cell Titer Glo® reagent, (Promega Corp., Madison, Wis.).Percent killing was determined as the ratio of signal from treatedversus untreated cells. The data in FIG. 5 suggest that IgM produced bythe 3B2 clone can elicit a potent CDC reaction on certain GD2 positivemelanoma cell lines.

Molecular analyses were performed to determine whether clones 3B2 and1470 were infected with EBV. Six PCR primer pairs ((EBNA2-1141f (SEQ IDNO: 45) and EBNA2-1440r (SEQ ID NO: 46); EBV2001f (SEQ ID NO: 47) andEBV2622r (SEQ ID NO: 48); EBV1901F (SEQ ID NO: 49) and EBV2822R (SEQ IDNO: 50); EBV169461f (SEQ ID NO: 51) and EBV170100r (SEQ ID NO: 52);EBV169480f (SEQ ID NO: 53) and EBV170080r (SEQ ID NO: 54); EBV8491F (SEQID NO: 55) and EBV9020r (SEQ ID NO: 56)) were designed to verify theexistence and intactness of the EBV genome. One of the primer pairsamplified the open reading frames of EBNA-2; two overlapping sets eachamplified the 5′ and 3′ end of the EBV genome; and one pair amplifiedthe origin of replication. Initially, both 3B2 and 1470 clones were EBVpositive as demonstrated by genomic PCR (data not shown). However,further subcloning by limiting dilution plating at less than one cellper well generated subclones of the 3B2 and 1470 lines that did notcontain the EBV genome, as determined by EBV-specific PCR analysis (datanot shown).

EBV-negative 3B2 subclones (AB527-HYB-3B2-EBVnull) have been placed withthe Amer. Type Cult. Coll. (10801 University Blvd., Manassas, Va.20110-2209) on Jul. 16, 2008 and have been assigned Accession No.PTA-9376. AB527-HYB-3B2-EBVnull subclones were subjected to a secondround of subcloning to identify clones that produced a single form ofimmunoglobulin J-chain, as the original hybridoma may produce human ormouse J-chain. To identify clones possessing a single form of J-chain,or J-chain-null clones, RT-PCR was performed using primers specific forhuman J-chain (hu-285F (SEQ ID NO: 57) and hu-418R (SEQ ID NO: 58)), ormouse J-chain (m-230F (SEQ ID NO: 59) and m-370R (SEQ ID NO: 60)). Theresults indicated that the 3B2 subclone, AB527-HYB-3B2-3C9, is EBV null,human J-chain null, murine J-chain positive and secretes approximately30 mg/L of monoclonal, GD2-specific IgM in static culture.

EXAMPLE 3 Production of a Transfectoma Cell Line Expressing GD2-SpecificIgM

Total RNA isolated from AB527-HYB-3B2-3C9, the EBV null, human J-chainnull, murine J-chain positive hybridoma, was reverse transcribed intocDNA and used as template for the PCR amplification of the IgM heavychain, using primers corresponding to SEQ ID NOs: 33 and 35, and lightchain, using primers corresponding to SEQ ID NOs: 36 and 38. The heavychain nucleotide sequence amplified from AB527-HYB-3B2-3C9 encodes apolypeptide having the amino acid sequence of SEQ ID NO: 40, and thelight chain nucleotide sequence amplified from AB527-HYB-3B2-3C9 encodesa polypeptide having the amino acid sequence of SEQ ID NO: 42. A secondround of PCR amplification was performed to add a human leader sequence,along with 5′ and 3′ restriction endonuclease recognition sites wereintroduced in order to facilitate cloning of the amplicons into pEE6.4(heavy chain) and pEE14.4 (light chain), both vectors are supplied aspart of Lonza Biologics Glutamine Synthetase Expression System (GSSystem). The second round PCR amplification of the IgM heavy chain wasconducted using primers corresponding to SEQ ID NOs: 34 and 35, whilethe light chain amplification used primers corresponding to SEQ ID NOs:37 and 38.

PCR amplified light chain cDNA was digested with HindIII and EcoRI andligated into similarly cut pEE14.4, while heavy chain cDNA was digestedwith HindIII and MfeI and ligated into pEE6.4 digested with HindIII andEcoRI (MfeI and EcoRI leave compatible “sticky” ends). Afterverification of the nucleotide sequence of the heavy and light chains,the heavy chain expression cassette was introduced in topEE14.4-AB527-LC as a NotI/PvuI fragment to construct the finalexpression vector pEE14.4-AB527-LC-HC (p0311) (FIG. 6). pEE14.4 is a 10Kb vector which contains a glutamine synthase mini gene, allowing forselection of stable transfectants in the presence of the GS inhibitorL-methionine sulfoximine (MSX). Transcription of the cDNA inserts inthis vector is controlled by the human CMV immediate early promoter,upstream of the cDNA insert is the hCMV-MIE 5′untranslated regionincluding introns 1, downstream of the cDNA insert is the SV40polyadenylation signal allowing for efficient polyadenylation of thetranscript (FIG. 7).

CHO cells were transfected with linearized, double-gene vector p0311 byelectroporation to allow for expression of recombinant GD2-specific IgM.Following transfection the cells were plated in 96-well plates usingnon-selective medium containing dialyzed fetal bovine serum (dFBS) andglutamine synthase supplement. The following day selective mediumcontaining dFBS, glutamine synthase supplement and MSX (10 μM finalconcentration) was added to each well, enabling selection of cellscontaining the expression vector. Only colonies perceived to have comefrom a single transfected cell were pursued.

Transfectants were screened for the presence of human IgM by ELISA.Colonies were expanded in serum-containing selection medium andsurviving colonies were examined by quantitative secondary assay toidentify the highest-producing colonies. The highest-producingtransfectants were adapted to chemically defined IS-CHO-CD™ medium(Irvine Scientific, Santa Ana, Calif.) and seeded at 2,500 cells/well inflat bottom 96 well plates (80 plates) in media containing 10 μM MSX.Drug-resistant colonies were analyzed for IgM production by ELISA.Colonies positive for IgM production were scaled up and analyzed forantibody productivity in subsequent secondary (6 well) and tertiary (20mL shake flask) assays. This resulted in the identification of anIgM-producing CHO-K1 SV transfectoma clone, 127C8. The 127C8 clone doesnot express J-chain, therefore, the recombinant AB527 IgM produced bythis cell line is J-chain-deficient. The amino acid sequences of theheavy and light chains of AB527 are represented by SEQ ID NOs: 40 and42, respectively.

EXAMPLE 4 AB527 Binds Specifically to GD2 In Vitro

Several experiments were conducted to determine whether recombinantAB527 could bind to purified GD2 in vitro. Initially, surface plasmonresonance experiments were performed to not only determine whetherrecombinant AB527 would bind to GD2, but also to determine whether itexhibited affinity for GM2 or GD3 (FIG. 8). The experiments wereperformed as follows: 40 μL of 0.3 mg/mL solutions of GD2 (BioDesign),GD3 (HyTest), and GM2 (USBiological) in 10 mM HEPES, 150 mM NaCl, 3 mMEDTA (HBS-E) containing 30% ethanol were injected over flow cells 2, 3,and 4, respectively, of a CM5 chip at a flow rate of 5 μl/minute using aBIAcore 3000 instrument. Five repeated injections of 20 μL 10 mM NaOHwere done following immobilization of each ganglioside until a stablebaseline was achieved. Next, 80 μL of 200 nM recombinant AB527 in HBS-Ebuffer was injected over all 4 flow cells (flow cell 1 was used as areference flow cell) at a flow rate of 20 μL/minute. Dissociation ofrecombinant AB527 was followed for 6 minutes. The chip surface wasregenerated with 50 μL of 101 mM NaOH.

To better characterize the binding kinetics of AB527, additional surfaceplasmon resonance experiments were conducted using serial dilutions ofAB527 and GD2 immobilized on a BIAcore CM5 chip. Briefly, two-foldserial dilutions of AB527, starting at 800 nM, were injected over aGD2-containing CM5 chip, described above, at a flow rate of 20 μL/minutein HBS-E buffer. The chip surface was regenerated between each cyclewith 50 μL of 10 mM NaOH. Equilibrium binding at each concentration(R_(eq)) was determined for each concentration tested in BIAevaluationsoftware and a plot of R_(eq) vs. concentration was then done. Theresulting data were fitted to a non-linear steady-state binding modelusing GraphPad Prizm® software. When the R_(eq) is plotted as a functionof AB527 concentration, the steady state K_(D), or the concentration ofAB527 at which one-half maximal binding is achieved, can be determined(FIG. 9). For AB527 binding to GD2, the steady state K_(D) value is4.5×10⁻⁹ M.

The specificity of recombinant AB527 was also tested by immunostainingvarious gangliosides separated by thin layer chromatography (TLC). Thefollowing gangliosides were used in the immunoTLC analysis: GM mix(Matreya, 0.5 mg/mL in ethanol contains GM1, GM2, and GM3), GD mix(Matreya, 0.5 mg/mL in ethanol, contains GD1a, GD1b, and GD3), GM4(Matreya, 1 mg/mL in ethanol), GD2 (BioDesign, 1 mg/mL in ethanol), andGM3 (USBiological, 1 mg/mL in ethanol). A total of 2 μl of eachganglioside was spotted on to a 5×10 cm silica TLC plate (EMD Chemicals,Inc.) and developed in a solution having a ratio of 58:37:8 of CHCl₃,MeOH, and 0.1M CaCl₂. TLC plates were stained for total ganglioside byspraying with 0.1% orcinol in 1.36NH₂SO₄ and heating at 100° C. ForimmunoTLC analysis, developed plates were first treated with 0.1%polyisobutylmethacrylate in H₂O, and then dried. The plates were thenblocked for 1 hour with 1% BSA in 1× phosphate-buffered saline (PBS).The blocked plate was then incubated in 2 μg/mL AB527 in 1×PBScontaining 1% BSA overnight at 4° C. The plates were subsequently washedwith 1×PBS, then incubated with goat anti-human IgG/IgM-horseradishperoxidase conjugate diluted 1:4000 in PBS containing 1% BSA. Plateswere washed, and bound antibody was detected by incubating the plates in0.3 mg/mL 4-chloro 1-napthol and 0.03% H₂O₂. The comparative stainingresults are shown in FIG. 10.

EXAMPLE 5 AB527 Labels GD2-Expressing Cells

The ability of AB527 to specifically label GD2-expressing melanoma celllines was assessed. GD2-positive lines: M14, M0023, M101, M18, M10-Vac,and PM0496, and GD2-negative lines: M21, M238, IMCD0023, MG1055 werecultured separately in RPMI1640 supplemented with 10% FBS, 2 mML-Glutamine, non-essential amino acids, and 6 μM HEPES and harvestedwith trypsin. Trypsinized cells were incubated with either 10 μg/mL ofcontrol IgM (human IgM, Pierce cat# 31146) (column I) or 10 μg/mL ofAB527 (column II) for 1 hour on ice. After 3 washes with 1×PBS, cellswere incubated with 10 μg/mL FITC-labeled goat anti human Ig (H+L)antibody (Southern Biotech, Birmingham, Ala.) for 1 hour on ice,followed by either flow cytometry (columns I and II, respectively) orlight microscopy and immunofluorescence microscopy analysis (columns IIIand IV, respectively). As shown in FIG. 11, AB527 labeled cells expressGD2 on their surface (column II). Conversely, none of the cell linesexamined showed staining with non-GD2-specific human IgM (column I).Immunofluorescent studies showed a direct correlation with flowcytometry data (column IV), light microscopy images are also shown forcomparative purposes (column III).

Cell-based ELISAs were performed to assess AB527 binding to theGD2-expressing cell lines M14, LLC-MK2, EL4 cells and to CHO-K1 cells(negative control), which provided the data to perform correspondingScatchard analyses. FIG. 12 provides a representative set of Scatcharddata for EL4 cells. AB527 binding to M14 and LLC-MK2 cells wasdetermined to be nearly identical (0.13±0.02 nM and 0.15±0.04 nM,respectively). Binding to EL4 cells was somewhat lower (0.93±0.16 nM).This difference may be due to the markedly lower expression of GD2observed on EL4 cells relative to M14 and LLC-MK2 (data not shown). Forthese experiments, M14, LLC-MK2, EL4, and CHO-K1 cells were obtainedfrom the American Tissue Type Collection (ATCC, Manassas, Va.). M14,EL4, and CHO-K1 cells were cultured in RPMI supplemented with 10% fetalbovine serum. LLC-MK2 cells were cultured in Medium 199 supplementedwith 1% horse serum. Cells were suspended in 1×PBS and added to a black,U-bottomed microtiter plate (Grenier cat. # 665209) at 2.5×10⁶cells/well, then pelleted at 1500 rpm for 5 minutes at room temperature.Supernatant was discarded, and cells were resuspended in a solutioncontaining AB527 starting at 20 μg/mL serially diluted 1:2 in PBScontaining 2% BSA. Cells were incubated with antibody for 1 hr at roomtemperature. Cells were washed three times with 200 μL PBS containing 2%BSA and 0.05% Tween®-20, centrifuging at 1500 rpm for 5 minutes tocollect cells between wash steps. Cells were then incubated in a PBSsolution containing 1 μg/mL horseradish peroxidase conjugated goatanti-human IgG/IgM (Jackson Immunoresearch) and 2% BSA for 1 hr at roomtemperature. Cells were washed as above. Bound AB527 was detected usingQuantaBlu™ fluorescent substrate (Pierce), with excitation/emissionwavelengths of 325 nm/420 nm, in a SpectraMax® M5 multimodal platereader (Molecular Devices). Raw data was plotted in SoftMax Pro® (ver.5.2) and fitted to a 4-parameter model. Data analysis was performed inGraphPad Prizm® software (ver. 4.03). Non-specific background was firstsubtracted (binding to CHO-K1 cells), and data were then analyzed byScatchard analysis.

Immunohistochemical (IHC) experiments were conducted to determinewhether AB527 exhibited cross-reactivity to melanoma cells that do notexpress GD2. Purified AB527, as well as a non-specific human IgM (Piercecat# 31146), were biotinylated using NHS ester coupling chemistry, at amolar ratio of 50:1 biotinylation reagent (EZ-Link™ sulfo-NHS-LC-biotin,Pierce cat# 21335) in phosphate-buffered saline, according to theprovided instructions, and used to stain various GD2-positive (M14) orGD2-negative (MG1055, RPM17951 and JS0592) cell lines. Briefly, eachmelanoma cell line was seeded on chamber slides and cultured overnightin RPMI1640 supplemented with 10% FBS, 2 mM L-Glutamine, non-essentialamino acids, and 6 μM HEPES. Cells were washed once with 1×PBS andformalin-fixed. Fixed cells were incubated overnight with 1 μg/mLbiotinylated AB527 or biotinylated nonspecific human IgM (negativecontrol) and labeled cells were detected using BioGenex Super Sensitive™Link-Label IHC Detection System. As shown in FIG. 13A, AB527specifically binds to GD2 positive cells and has minimal crossreactivity with GD2 negative cells, while the non-specific isotopecontrol antibody does not stain either cell type (FIG. 13B).

Binding characteristics of AB527 were also assessed using tumor andnormal tissue samples. AB527 and a nonspecific normal human IgM (hIgM)were biotinylated at Covance Research Products and used for IHC studiesat Charles River Laboratories. Human melanoma cancer cells, M14, wereimplanted in nude rats to establish xenograft tumors, which were thenused as GD2 positive control tissue for IHC method development.Preliminary studies using a direct avidin-biotin-peroxidase complex(ABC) procedure yielded positive staining of M14 tumor sections withbiotinylated AB527 (FIG. 14( b)) but not with biotinylated hIgM (FIG.14( a)). As expected, lymphocytes in cryosections of normal human spleendid not stain with either antibody. After titration of the antibodies,the staining concentrations for biotinylated AB527 and biotinylated hIgMselected for the GLP human tissue cross-reactivity study were set at 3and 15 μg/mL. Using this method, a study was conducted to determineAB527 binding in 35 normal human tissues (3 samples/donors per tissue).As expected, high affinity membranous staining (positive cell membranestaining at the lower concentration of AB527-3 μg/mL) could be detectedin M14 tumor sections. As shown in table 2, high affinity membranousstaining was only found in the epithelial cells of esophagus (2 of 3samples), fallopian tube (1 of 3 samples), and ureter (1 of 3 samples),as well as in decidual cells of placenta (3 of 3 samples), and reticularcells of thymus (2 of 3 samples). All the other tissues showed no highaffinity membranous staining (table 2).

TABLE 2 AB527 Immunohistochemistry in Normal Human TissuesPositive/Total Positive/Total Positive/Total Tissue (Cell Type) Tissue(Cell Type) Tissue (Cell Type) Adrenal Negative Liver Negative SkinNegative Blood Cells Negative Lung Negative Spinal Cord Negative BloodVessels Negative Lymph Node Negative Spleen Negative Bone MarrowNegative Ovary Negative Striated Muscle Negative Brain (Cortex) NegativeFallopian Tube 1/3 Testis Negative (Epithelium) Brain Negative PancreasNegative Thymus 2/3 (Cerebellum) (Reticular Cells) Breast NegativeParathyroid Negative Thyroid Negative Eye Negative Peripheral NegativeTonsil Negative Nerve Esophagus 2/3 Pituitary Negative Ureter 1/3Epithelium (Epithelium) GI Tract Negative Placenta 3/3 Urinary Negative(Decidual Cells) Bladder Heart Negative Prostate Negative Uterus (Body)Negative Kidney Negative Salivary Gland Negative Uterus (Cervix)Negative

EXAMPLE 6

AB527 Facilitates Complement Dependent Cytotoxicity of Cells ExpressingGD2

To determine whether AB527 mediates CDC, the melanoma cell linesdescribed above for FIG. 11 were incubated with partially purifiedantibody from the AB527-HYB-3B2-3C9 clone or AB527. Target cells werecultured in RPM11640 supplemented with 10% FBS, 2 mM L-Glutamine,non-essential amino acids, and 6 μM HEPES and harvested with trypsinbefore use. Target cells were incubated with 10 μg/mL of either AB527 orAB527-HYB-3B2-3C9 clone IgM in the presence of 20% human serum. Afterincubation for 1 hour at 37° C., live cells were identified with CellTiter Glo® reagent (Promega Corp., Madison, Wis.). Percent killing wasdetermined as the ratio of signal from treated versus untreated cells.The data in FIG. 15 show that both antibodies can elicit a potent CDCreaction on certain melanoma cell lines, with AB527 consistently causinga greater degree of CDC. The results of the experiments using AB527described in this Example are summarized in table 3.

TABLE 3 Comparison of FACS, Immunofluorescence Microscopy and CDCanalysis of human melanoma cell lines treated with AB527. AB527 FACSAB527-mediated Cell Line Staining AB527 IHC CDC Killing M14 + + +LF0023 + + + M101 + + + M18 + + + M10VAC + + + M21 +/− − − M238 − − −PM0496 + + − IMCD0023 +/− − − MG1055 − − −

Additional work was done to show that AB527 facilitates CDC only ofcells that express GD2 in the presence of complement. As shown in FIG.16, AB527-mediated CDC activity only occurred to appreciable levels inthe presence of complement and GD2-expressing cells, such as M14 cells,as opposed to 1205LU cells, which are human tumor cells that do notexpress GD2.

To assess the in vitro ability of IgG1 isotype-switched AB527 to induceCDC, the HindIII/BamHI variable region DNA fragment of AB527 was clonedin-frame into a HindIII/BamHI digested pEE6.4 vector containing the IgG1constant region. Co-expression with the AB527 light chain caused theproduction of an IgG1 version of AB527. The ability of this antibody tomediate CDC of M14 cells was compared to the IgM version of AB527 at1-100 μg/mL, as shown in FIG. 17.

Studies were also conducted to assess the role that J-chain might playin AB527-mediated CDC. To produce AB527 IgM molecules having J-chain,plasmid p0362, which encodes J-chain, was transfected into CHOK1-SVcells and a stable, J-chain-expressing clonal line was generated. TheAB527 expression plasmid was then introduced into this line to generateclones expressing AB527 and J-chain. Three clones named 5.F2-JC,5.F8-JC, and 6.C3-JC were isolated. The antibodies were purified inparallel to compare their relative CDC activity against a non-J-chaincontaining version of AB527. Dose response curves are shown in FIG. 18.The calculated 90% of effective dose (ED90) for AB527, 5.F2-JC, 5.F8-JC,and 6.C3-JC was 1.21, 4.35, 2.52, and 6.81 μg/mL, respectively. IgMcontaining J-chain (5.F2-JC, 5.F8-C, and 6.C3-JC) appeared to be lesseffective at mediating CDC compared to AB527 without J-chain.

EXAMPLE 7 Capture of IgM from Conditioned Culture Supernatant by ProteinA Affinity Chromatography

A column was packed with CPG3000A-protein A resin (Millipore). Thevolume of resin used to capture IgM was estimated, with theunderstanding that 1 mL of resin binds approximately 8 mg of IgM at aflow rate of 76 cm/h (table 4). Once the column was loaded with thedesired amount of resin, it was connected to an FPLC apparatus andwashed with 5 column volumes of purified water; 3 column volumes of 20mM HCl, pH 1.5 (buffer A2); and 3 column volumes of 6 M guanidine-HCl(buffer A3). Before loading sample, the column was equilibrated with 5column volumes of a 10 mM sodium phosphate buffer containing 200 mM NaCland 0.01% Tween®-80, pH 7.5 (buffer A1).

Conditioned culture supernatant (CCS) was clarified by filtrationthrough a 0.22 μm membrane filter. Detergent was added to the filteredCCS resulting in a solution containing 1% Triton® X-100 and 0.1% TNBPand incubated at 4° C. for more than 2 hours. Detergent-treated CCS wasapplied to an equilibrated CPG3000A protein A column and processed at aflow rate of 76 cm/h. After washing away unbound proteins with 10 columnvolumes of buffer A1, IgM was eluted from the column using a 5 mM sodiumphosphate buffer supplemented with 3 M MgCl₂, pH 6.8 (buffer B).Remaining bound proteins were removed from the column by applying 3column volumes of buffer A2 and 3 column volumes of buffer A3. To assesspurity, a reducing SDS-PAGE gel comparing detergent-treated CCS appliedto the protein A column (Load) and AB527 collected from pooled fractionsafter elution (Elute) was performed (FIG. 19—arrows point to the IgM μchain (˜70 kD) and light chain (˜25 kD)). Protein A affinitychromatography allowed for recovery of ≧90% of input IgM, which issubstantially free of contaminants.

TABLE 4 Dynamic binding capacity of IgM by CPG3000A protein A resin.Varying volumes of CCS containing AB527 were run through a 10 × 25 mmCPG3000A column at different linear flow rates. The concentration ofAB527 loaded into the column and present in the flowthrough weremeasured by determining their relative concentrations as compared tothat of a standard curve for known IgM standards determined on a proteinA column (POROS A, Applied Biosystems), fitted on a HPLC system. Theratio of AB527 in each flowthrough to the amount of loaded antibody wascomputed for each injection to determine the dynamic binding capacity ofthe column at 5% and 10% IgM breakthrough. Dynamic Binding CapacityLinear (DBC)¹ AB527 in Flow (mg AB527/L CPG3000A) Flowthrough (FT) (mg)Rate 5% 10% 5% Break- 10% (cm · hr⁻¹) Breakthrough Breakthrough throughBreakthrough 23 5780 6420 11559 12840 38 6869 7630 13737 15260 76 43955437 8789 10873 152 3126 3910 6252 7819 ¹DBC was determined on a 1.0 cm× 2.5 cm bed (2 ml bed volume)

EXAMPLE 8 Separation of IgM from Conditioned Culture SupernatantContaminants by Cation Exchange Chromatography

To remove contaminants, IgM suspended in buffer B was concentrated bydiafiltration using a Prep/Scale-TFF 1 cartridge (Millipore) and a10-fold volume of a buffer containing 10 mM sodium phosphate, 75 mMNaCl. Diafiltration was conducted at a flow rate of approximately 200mL/minute, with inlet pressure kept at less than 20 PSI and permeateflow at approximately 60 mL/minute. A solution of 10% Triton® X-100 wasadded to the diafiltered sample to yield a 1% final concentration ofTriton® X-100, and the sample was incubated at room temperature for 1hour and filtered through a 0.2 μm membrane.

A cation exchange column was packed with MacroCap™ SP resin (GEHealthcare). The volume of resin used to capture IgM was estimated, withthe understanding that 1 mL of resin binds approximately 9 mg of IgM ata flow rate of 76 cm/h (FIG. 20). Once the column was loaded with thedesired amount of resin, it was connected to an FPLC apparatus andwashed with 2 column volumes of purified water; 3 column volumes of 0.5M NaOH (buffer A6), and 3 column volumes of 2 M NaCl. Before loadingsample, the column was equilibrated with 5 column volumes of a buffercontaining 10 mM sodium phosphate, 75 mM NaCl, 0.01% Tween®-80, pH 6.8(buffer A4).

The diafiltered sample was applied to the column and processed at a flowrate of 76 cm/h. After washing away unbound proteins with 5 columnvolumes of buffer A4, IgM was eluted from the column using 4 columnvolumes of a 10 mM sodium phosphate buffer containing 200 mM NaCl and0.01% Tween®-80, pH 6.8 (buffer C). Remaining bound proteins wereremoved from the column by applying 3 column volumes of buffer A6 and 3column volumes of 2 M NaCl As shown in FIGS. 13A and B, the flowthroughmaterial largely contained a mixture of IgM trimers and dimers (FIG.21A); however, the eluted material was almost exclusively pentameric IgM(FIG. 21B). Cation exchange chromatography provides ≧95% yield ofpentameric IgM that is substantially free of contaminants andincompletely assembled IgM (FIG. 22).

EXAMPLE 9 Separation of 12M from Conditioned Culture SupernatantContaminants by Hydroxyapatite Chromatography

The hydroxyapatite column was prepared using CHT® II CeramicHydroxyapatite, 80 μm bead size (Bio-Rad Laboratories, #157-8100). Thevolume of resin used to capture IgM was estimated, with theunderstanding that 1 mL of resin binds approximately 20 mg of IgM at aflow rate of 76 cm/h, and at this flow rate there is less than 5% lossof IgM in the column flow-through. The column was packed by hydratingthe matrix with 200 mM potassium phosphate pH 9.0, using 0.54 g of drymatrix for each mL of desired column-bed volume. The hydrated matrix wasadded to the column, allowing it to settle before the resin wasequilibrated with 5 column volumes of a buffer containing 10 mM sodiumphosphate, 100 mM NaCl, 0.01% Tween®-80, pH 6.8 (buffer A8)

To reduce contaminants, IgM suspended in 10 mM sodium phosphate, 200 mMNaCl, 0.01% Tween®-80, pH 6.8 was diluted with an equivalent volume of0.01% Tween®-80 and added to the equilibrated column. The IgM suspensionwas processed through the column at a flow rate of 76 cm/h. The columnwas washed with 10 column volumes of buffer A8, to remove unboundsubstances. Bound IgM was eluted with 4 column volumes of a 175 mMsodium phosphate buffer containing 100 mM NaCl and 0.01% Tween®-80, pH6.8 (data not shown). Following elution, remaining bound proteins wereremoved from the column with a 500 mM sodium phosphate buffer containing100 mM NaCl, pH 6.8. The resulting IgM solution was substantially freeof contaminants. The solution eluted from the hydroxyapatite matrix,when preceded by protein A affinity chromatography and cation exchangechromatography, described herein, was over 99% pure as compared to theclarified cell culture supernatant.

EXAMPLE 10 Production of a Ganglioside-BSA Conjugates

A GD2-BSA conjugate was produced to provide an effective GD2 antigen toexamine the specificity of GD2 specific antibodies by immunoassay. Inaddition, GM2-BSA and GM3-BSA conjugates were produced to serve ascontrols for GD2-BSA binding specificity. The covalent conjugates wereprepared by site-specific reductive amination of the gangliosides byamines present on BSA, utilizing sodium cyanoborohydride as a mildreducing agent. Coupling is thought to occur mainly to terminal ε-aminogroups on the side chains of lysine residues present in BSA.

To prepare the conjugates, 400 μl of a 2 mg/ml solution of BSA dissolvedin 0.05 M carbonate-bicarbonate buffer, pH 9.6, was combined with 500 μlof a 1 mg/ml solution of GD2 (Biodesign International, #A86168H), GM2(Axxora, #ALX-302-005-M001), or GM3 (Axxora, #ALX-302-003-M002)dissolved in absolute ethanol; 2070 μl of 0.05 M carbonate-bicarbonatebuffer, pH 9.6; and 30 μl of a 5 M sodium cyanoborohydride solution of10 mM sodium hydroxide. The reaction mixture was incubated at 25° C. fortwo hours before adding 1200 μl of 1M ethanolamine, pH 8.0 andincubating at 25° C. for an additional 15 minutes. The reaction solutionwas placed in dialysis tubing (Pierce Biotechnology, # 68100) anddialyzed twice in 1 L of a 50 mM sodium carbonate-bicarbonate solution,pH 9.6, at 2-8° C. for 6-18 hours.

EXAMPLE 11 AB527 Binds to BSA-Conjugated GD2

Antigenicity of the GD2-BSA conjugate was assessed by ELISA. ELISAcoating buffer was prepared by dissolving one carbonate/bicarbonatecapsule (Sigma) in 100 mL ultrapure water. GD2-BSA conjugate was dilutedto a concentration of 2 μg/ml, in ELISA coating buffer, and added towells of a 96-well ELISA plate (Greiner Bio-One cat #655081). The plateswere sealed and incubated at 2-8° C. for 16-24 hours. Followingincubation, the coating buffer was removed from the wells and 0.3mL/well of ELISA blocking solution (PBS supplemented with 2.5% BSA (w/v)and 0.05% Tween®-20) was added. Plates were incubated, while shaking ona microplate shaker, for 2 hours at 21-25° C. before adding AB527antibody. Two-fold serial dilutions of AB527 (initial workingconcentration of 2 μg/ml) were added to the blocked ELISA plates andincubated with shaking on a microplate shaker at 21-25° C. for 1 hour.Plates were washed three times on the Dynex Ultrawash™ plate washer,using 0.3 mL/well ELISA wash buffer. Bound AB527 was detected withHRP-conjugated goat anti-human IgG+IgM (H+L), diluted 1:10,000 in ELISAblocking buffer. The HRP-conjugate was removed and wells were washedthree times with 0.3 mL/well ELISA wash buffer. SureBlue™ TMB substrate(50 μl/well) was added to washed wells and plates were incubated at21-25° C. for 15 minutes, before AB527 binding was assessed by lightabsorption (450 nm) (FIG. 23). Nonspecific absorption at 450 nm wasdetermined to be 0.065 in the absence of AB527.

In contrast to the GD2-BSA conjugate, AB527 did not recognize otherganglioside-BSA conjugates. The gangliosides GM2 and GM3 were conjugatedto BSA, by the method described for GD2, and both were assessed, byELISA, for the ability to elicit AB527 binding. As shown in FIG. 24,AB527 recognized only the GD2-BSA conjugate. It should be noted that aGM2-specific antibody was able to detect the GM2-BSA conjugate; however,an appropriate GM3-specific antibody was not available to assess theantigenic nature of the GM3-BSA conjugate (data not shown).

EXAMPLE 12 Effect of CMS on AB527-Expressing Cells Grown in GIBCO-CD CHOComplete Medium

To determine the effect of culture media supplement (CMS) on cell growthand antibody production, cells were revived and cultured in a shakeflask in 20 mL GIBCO-CD-CHO complete medium. All cultures were incubatedin 125 mL shake flask at 37° C., 5% CO₂, 120 rpm. The seeding densitywas 3.5×10⁵ cells/mL at >95% viability. Cells were cultured for 14 daysin either CD-CHO media or in CD-CHO media supplemented on days 3-7 with2% (vol/vol) CMS. Following addition of CMS on day 7 a total of 10% ofthe initial culture volume had been supplemented with CMS. Viable celldensity (FIG. 25), percent viability (FIG. 26), and integral viable celldensity (IVC) (FIG. 27) were determined using a CEDEX automated cellcounter on days 0, 3, 5, 7, 10, 13, and 14. Antibody production wasdetermined for each group of cultured cells on day 14 by protein A HPLC(FIG. 28).

As shown in FIG. 25, cultured cells provided with 2% CMS from day 3 today 7 achieved a maximum viable cell density of 12.8×10⁶ cells/mL on day7. The control cell culture, without CMS, reached a maximum cell densityof 10.7×10⁶ cells/mL on day 7. The use of CMS replenishes limitingmedium components such as glucose, amino acids and vitamins and helpsthe culture reach a higher cell density. In both cell cultureconditions, cells maintained a good viability (>90%) until day 7 (FIG.26); however, cultured cells provided CMS from day 3 to day 7demonstrated a better viability than the control culture after day 7. Itis possible that some key nutrients were depleted in the control cultureafter day 7 while the CMS added back those key nutrients and helped thecell maintain a better viability.

On day 14, total antibody production was assessed for each culture.Cells cultured with CMS reached a maximum antibody titer of 7240 mg/L,much higher than that observed for the control culture (3036 mg/L) (FIG.28). The higher antibody titer could be a result of a higher specificantibody production rate after the addition of the nutrients.Investigation of the cell growth and antibody production profiles foreach growth condition demonstrated that the culture provided CMSachieved a 140% increase in antibody production (table 5). Even thoughthe doubling time was the same for both cultures, the CMS did helpcultures achieve a 50% improvement in IVC and another 50% increase inspecific antibody production rate. The 50% improvement in IVC was due toa higher maximum cell density achieved in the culture with CMS. Sincethe final antibody titer is equal to the IVC multiple specificproduction rate, it suggested that both IVC and specific antibodyproduction rate contributed to the 140% increase in antibody productionin the cell culture with CMS. Table 5 summarizes the effects the CMS onAB527-expressing cells.

TABLE 5 Summary of the Effects of CMS on Cell Growth and AntibodyProduction of AB527- Expressing Cells. Specific Antibody MaximumIntegral of Viable Cell Production Rate Culture Viable Cell FinalAntibody Doubling Density (IVC) (Qp) Conditions Density (10⁵/mL) Titer(mg/L) Time (DT) (cells · day/mL) (mg/10⁹ cells/day) CD-CHO 107.00 303624.17 6.85E+7 44.08 Control CMS 128.40 7239.9 24.17 1.04E+8 66.95

EXAMPLE 13 Effect of Valeric Acid on AB527-Expressing Cells Grown inGIBCO-CD CHO Complete Medium

To determine the effect of valeric acid on cell growth, AB527-expressingcells were cultured in GIBCO-CD-CHO complete medium supplemented withvaleric acid to a concentration of 0 mM, 1 mM, 2 mM, 4 mM, or 8 mM.Cells were cultured for 14 days and viable cell density, percentviability, and integral viable cell density were determined using aCEDEX automated cell counter on days 0, 3, 5, 7, 10, 13, and 14.Antibody production was determined for each group of cultured cells onday 14 by protein A HPLC.

Cells cultured without valeric acid achieved the maximum cell density of10.8×10⁶ cells/mL at day 7; however, cells cultured with valeric acidgrew much slower as the concentration of valeric acid increased. Forexample, cells cultured in 8 mM valeric acid, reached a maximum celldensity of 5.4×10⁶ cells/mL at day 7, which is the lowest maximum celldensity of all cultures. Cell viability studies demonstrated that cellscultured with valeric acid had lower viability before day 7, with thelowest viability at day 7 (67.4%) observed for cells cultured in 8 mMvaleric acid. These results indicate that higher concentration of thevaleric acid inhibited cell proliferation. After day 7, the viability ofthe control culture dropped much quicker; however, this was likely dueto consumption of key nutrients since this cell culture had a muchhigher cell density than those cultured in valeric acid (table 6).

Unexpectedly, however, valeric acid had a positive effect on antibodyproduction. All cells cultured in the presence of valeric acid, withexception of the highest concentration of 8 mM, had higher antibodytiters than the control culture at day 14. Cell cultured in 1 mM valericacid produced the highest antibody titer (4137 mg/L) at day 14, which is36% higher than that of the control culture (3036 mg/L) (table 6).

Even though the valeric acid inhibited the cell growth, it did notdemonstrate the significant negative effect on the integral of viablecell density, except at the highest concentration of 8 mM. The IVCvalues at day 14 were quite similar for the other four cultureconditions. It is likely that the inhibition of valeric acid on the cellproliferation led to a slower consumption of the nutrients, thusavoiding late-stage nutrient-depleted media, which was encountered bythe control culture.

In summary, the cell culture can achieve a better antibody production inpresence of the valeric acid. The specific antibody production rateranging from 52 to 58 mg/10⁹ cells/day was observed in cultures withvaleric acid (table 6), which are much higher than the value of 44mg/10⁹ cells/day observed for the control culture. Thus, the highestantibody production achieved by cells cultured in 1 mM valeric acid wasmainly due to the increases in the specific antibody production (table6).

TABLE 6 Summary of the Effects of Valeric Acid on Cell Growth andAntibody Production of AB527-Expressing Cells. Maximum Specific AntibodyViable Cell Integral of Viable Cell Production Rate Culture DensityFinal Antibody Doubling Density (IVC) (Qp) Conditions (10⁵/mL) Titer(mg/L) Time (DT) (cells · day/mL) (mg/10⁹ cells/day) CD-CHO 107.00 303624.17 6.85E+7 44.08 Control Valeric Acid 94.60 4137.1 24.17 6.74E+758.44 (1 mM) Valeric Acid 81.00 3706.7 24.17 6.55E+7 54.45 (2 mM)Valeric Acid 77.40 3600.1 24.17 6.72E+7 52.24 (4 mM) Valeric Acid 54.402760.4 24.17 4.76E+7 56.16 (8 mM)

1. An isolated polynucleotide encoding an antibody, or antigen bindingfragment thereof, that specifically binds to human GD2, having a heavychain CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a heavychain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, a heavychain CDR3 comprising the amino acid sequence of SEQ ID NO: 12, a lightchain CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a lightchain CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and alight chain CDR3 comprising the amino acid sequence of SEQ ID NO:
 28. 2.The isolated polynucleotide of claim 1, comprising the nucleic acidsequences of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18,SEQ ID NO: 19, and SEQ ID NO:
 20. 3. The isolated polynucleotide ofclaim 1, comprising the nucleic acid sequences of SEQ ID NO: 8 and SEQID NO:
 24. 4. The isolated polynucleotide of claim 1, comprising thenucleic acid sequences of SEQ ID NO: 39 and SEQ ID NO:
 41. 5. A vectorcomprising the isolated polynucleotide of claim
 1. 6. An isolated cellcomprising the vector of claim
 5. 7. The isolated cell of claim 6,wherein the cell is a bacterial cell.
 8. The isolated cell of claim 6,wherein the cell is a eukaryotic cell.
 9. The isolated eukaryotic cellof claim 8, wherein the cell is a Chinese Hamster Ovary (CHO) cell. 10.The isolated eukaryotic cell of claim 8, wherein the cell does notexpress J-chain.
 11. The isolated eukaryotic cell of claim 10, whereinthe cell produces a pentameric IgM antibody.
 12. The isolated eukaryoticcell of claim 10, wherein the cell produces more pentameric IgM thanhexameric IgM.
 13. A method of making an antibody or antigen-bindingfragment thereof that specifically binds to human GD2 and includes aheavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 10, aheavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, aheavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 12, alight chain CDR1 comprising the amino acid sequence of SEQ ID NO: 26, alight chain CDR2 comprising the amino acid sequence of SEQ ID NO: 27,and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:28, comprising culturing the isolated eukaryotic cell of claim 10 underconditions suitable to produce the antibody or antigen-binding fragmentthereof and recovering the antibody or antigen-binding fragment thereoffrom the cell culture.
 14. A method of making an antibody orantigen-binding fragment thereof that specifically binds to human GD2and includes a heavy chain CDR1 comprising the amino acid sequence ofSEQ ID NO: 10, a heavy chain CDR2 comprising the amino acid sequence ofSEQ ID NO: 11, a heavy chain CDR3 comprising the amino acid sequence ofSEQ ID NO: 12, a light chain CDR1 comprising the amino acid sequence ofSEQ ID NO: 26, a light chain CDR2 comprising the amino acid sequence ofSEQ ID NO: 27, and a light chain CDR3 comprising the amino acid sequenceof SEQ ID NO: 28, comprising culturing the isolated cell of claim 6under conditions suitable to produce the antibody or antigen-bindingfragment thereof and recovering the antibody or antigen-binding fragmentthereof from the cell culture.
 15. A recombinant polynucleotide encodingan antibody heavy chain, or a fragment thereof, of an antibody thatspecifically binds to human GD2, wherein the heavy chain CDR1 of theantibody comprises the amino acid sequence of SEQ ID NO: 10, the heavychain CDR2 of the antibody comprises the amino acid sequence of SEQ IDNO: 11, and the heavy chain CDR3 of the antibody comprises the aminoacid sequence of SEQ ID NO: 12, wherein the encoded heavy chain orfragment thereof comprises SEQ ID NOs: 10, 11, and
 12. 16. Therecombinant polynucleotide of claim 15, comprising the nucleic acidsequences of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO:
 4. 17. Therecombinant polynucleotide of claim 15, comprising the nucleic acidsequence of SEQ ID NO:
 8. 18. The recombinant polynucleotide of claim15, comprising the nucleic acid sequence of SEQ ID NO:
 39. 19. A vectorcomprising the recombinant polynucleotide of claim
 15. 20. An isolatedcell comprising the vector of claim
 19. 21. The isolated cell of claim20, wherein the cell is a bacterial cell.
 22. The isolated cell of claim20, wherein the cell is a eukaryotic cell.
 23. The isolated eukaryoticcell of claim 22, wherein the cell is a Chinese Hamster Ovary (CHO)cell.
 24. An isolated cell comprising a vector having a recombinantpolynucleotide encoding an antibody heavy chain, or a fragment thereof,of an antibody that specifically binds to human GD2, wherein the encodedheavy chain or fragment thereof includes a heavy chain CDR1 comprisingthe amino acid sequence of SEQ ID NO:10, a heavy chain CDR2 comprisingthe amino acid sequence of SEQ ID NO:11, and a heavy chain CDR3comprising the amino acid sequence of SEQ ID NO:12, the isolated cellfurther comprising a separate vector having a recombinant polynucleotideencoding an antibody light chain, or a fragment thereof, of an antibodythat specifically binds to human GD2, wherein the encoded light chain orfragment thereof includes a light chain CDR1 comprising the amino acidsequence of SEQ ID NO:26, a light chain CDR2 comprising the amino acidsequence of SEQ ID NO:27, and a light chain CDR3 comprising the aminoacid sequence of SEQ ID NO:28.
 25. A method of making an antibody orantigen-binding fragment thereof that specifically binds to human GD2and includes a heavy chain CDR1 comprising the amino acid sequence ofSEQ ID NO: 10, a heavy chain CDR2 comprising the amino acid sequence ofSEQ ID NO: 11, a heavy chain CDR3 comprising the amino acid sequence ofSEQ ID NO: 12, a light chain CDR1 comprising the amino acid sequence ofSEQ ID NO: 26, a light chain CDR2 comprising the amino acid sequence ofSEQ ID NO: 27, and a light chain CDR3 comprising the amino acid sequenceof SEQ ID NO: 28, comprising culturing the isolated cell of claim 24under conditions suitable to produce the antibody or antigen-bindingfragment thereof and recovering the antibody or antigen-binding fragmentthereof from the cell culture.
 26. The method of claim 25, wherein theisolated cell is a eukaryotic cell that does not express J-chain. 27.The method of claim 26, wherein the antibody is a recombinant pentamericIgM antibody.
 28. The method of claim 26, wherein the isolated cellproduces more pentameric IgM than hexameric IgM.